Replication protein A and use

ABSTRACT

Methods and compositions for modulating DNA metabolism are provided. Nucleotide and amino acid sequences encoding a maize replication protein A subunits are provided. The sequences can be used in expression cassettes for modulating DNA replication, DNA repair, and recombination.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of co-pending U.S. applicationSer. No. 09/396,149 filed Dec. 15, 1999, and claims the benefit of U.S.Provisional Application Serial No. 60/100,690 filed Sep. 17, 1998, nowabandoned and U.S. Provisional Application Serial No. 60/123,896 filedMar. 11, 1999, now abandoned, which are all herein incorporated byreference.

FIELD OF THE INVENTION

[0002] The invention relates to the genetic manipulation of plants,particularly to modulating DNA metabolism in transformed plants andplant cells.

BACKGROUND OF THE INVENTION

[0003] Replication protein A (RPA) is a single-stranded DNA-bindingprotein that is required for multiple processes in eukaryotic cells. RPAfrom human cells is a stable complex of 70-, 32-, and 14-kDa subunits.Homologues of RPA have been identified in all eukaryotes examined.However, only human RPA and closely related homologues can support SV40DNA replication.

[0004] The RPA complex appears to be highly conserved in all eukaryotes.The three RPA genes in budding yeast cells are essential for cellviability. Nevertheless, yeast RPA only partially substitutes for humanRPA in the in vitro replication of simian virus 40 indicating thatspecies-specific interactions between RPA and other replication proteinsmay be important for its biological activity.

[0005] RPA binds tightly to single stranded DNA as a heterotrimericcomplex. The binding activity has been localized to the 70 kDa subunit.The affinity of RPA for both double-stranded DNA and RNA is at leastthree orders of magnitude lower than it is for single-stranded DNA. Ithas been reported that RPA binds preferentially to the pyrimidine-richstrand of both S. cerevisiae sequences and the SV40 origin ofreplication. However, studies examining the determinants of replicationorigins in S. cerevisiae indicate that this preferential binding is notcritical for the initiation of DNA replication.

[0006] Subunits of RPA in the 70-, 32- and 14 kDa ranges have beenidentified from various sources. The 32 kDa subunit has also beenreferred to as “RPA2”, “B”, “small”, “32 kDa”, “P32”, “P34”, and“middle” subunit. For the purposes of this invention, the “middle”subunit is intended as the subunit having a molecular weight of about 32kDa.

[0007] The middle subunit of RPA has a role in cell cycle regulation;single stranded DNA binding; affinity of DNA binding;species-specificity of DNA binding; DNA recombination, repair,replication and metabolism; and response to DNA damages. (Anderson(1966) Calif. Inst. Technol.; Seroussi et al. (1993) J. Biol. Chem.268:7147-54; Kenny et al. (1989) Proc. Natl. Acad. Sci. USA 86:9757-61;Brush et al. (1995) Methods Enzymol. 262:522-48; Stigger et al. (1994)Proc. Natl. Acad. Sci. USA 91:579-83; Philipova et al. (1996) Genes Dev.10:2222-33).

[0008] Much research has centered on the exploration of the biochemicaland genetic mechanisms by which cell cycle regulation of DNA synthesisis achieved. While there have been advances in delineating the existenceof cell cycle proteins, more information is needed on the mechanism ofaction of DNA replication, recombination, and repair. Furthermore,methods for regulating or altering the cell cycle is needed.

[0009] Related Literature

[0010] Braun et al. (1997) Biochemistry 36:8443-8454; report on the roleof protein-protein interactions and the function of replication proteinA. It is reported that RPA modulates the activity of DNA polymerase a bymultiple mechanisms.

[0011] Loor et al. (1997) Nucleic Acids Research 25:5041-5046 report onthe identification of DNA replication in cell cycle proteins thatinteract with proliferating cell nuclear antigen.

[0012] Longhese et al. (1994) Molecular and Cellular Biology14:7884-7890 report that replication factor A is required for in vivoDNA replication, repair, and recombination.

[0013] Stigger et al. (1998) J. Biol. Chem. 273:9337-9343 provide afunctional analysis of human replication protein A in nucleotideexcision repair.

[0014] Abremova et al. (1997) Proc. Natl. Acad. Sci. USA 94:7186-7191report that the interaction between replication protein A and p53 isdisrupted after ultraviolet damage in a DNA repair-dependent manner.

[0015] New et al. (1998) Nature 391:407-410 reports that RAD52 proteinstimulates DNA strand exchange by RAD51 and replication protein A.Stimulation was dependent on the concerted action of both RAD51 proteinand RPA implying that specific protein-protein interactions betweenRAD52 protein, RAD51 protein and RPA are required.

[0016] Dutta et al. (1992) EMBO J. 11(6): 2189-2199 and Niu et al.(1997) J. Biol. Chem. 272(19):12634-41 report cell cycle-dependentphosphorylation of the middle subunit of RPA, implying a role for thesubunit in cell cycle regulation.

[0017] Bochkareva et al. (1998) J. Biol. Chem. 273(7):3932-3936 reportthe formation of a single stranded DNA binding site on the human RPAmiddle subunit.

[0018] Mass et al. (1998) Mol Cell. Biol. 18(11):6399-6407 report thatthe RPA middle subunit contacts nascent simian virus 40 DNA,particularly the early DNA chain intermediates synthesized by DNApolymerase alpha-primase (RNA-DNA primers), but not more advancedproducts.

[0019] Lavrik et al. (1998) Nucleic Acids Res 26(2):602-607 report onlocation of binding of individual subunits of human RPA to DNAprimer-template complexes in various elongation reactions.

[0020] Sibenaller et al. (1998) 37(36):12496-12506 report thatdifferences in the activity of the middle (32 kDa) and the small (14Kda) subunits of RPA are responsible for variations in the singlestranded DNA-binding properties of Saccharomyces cerevisiae and humanRPA, thus implying a role for the subunits in species-specificity of DNAbinding of RPA.

SUMMARY OF THE INVENTION

[0021] Compositions and methods for modulating DNA metabolism in a hostcell is provided. Particularly, the complete cDNA and amino acidsequence for homologues of maize replication protein A (RPA) large- andmiddle subunits are provided. The sequences of the invention find use inmodulating DNA replication, DNA repair, and recombination.

[0022] Transformed plants can be obtained having altered metabolicstates. The invention has implications in genetic transformation andgene targeting in plants. Additionally, the methods can be used topromote cell death particularly in an inducible or tissue-preferredmanner.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023]FIG. 1 provides a comparison of eukaryotic RPA large subunit aminoacid sequences. Amino acid sequences for the RPA large subunits fromSaccharomyces cerevisiae (Rfal Yeast, SEQ ID NO:10), Schizosaccharomycespombe (Rfal_Schpo, SEQ ID NO: 9), Drosophila melanogaster(Rfal_Drome,SEQ ID NO:8), Homo sapiens (Rfal_Human, SEQ ID NO: 7), Xenopus laevis(Rfa_Xenla, SEQ ID NO: 6), and Oryza sativa (024183, SEQ ID NO:5) werecompared with the maize RPA LS homologue 1 (ZMRPALSH1, SEQ ID NO:2) andhomologue 2 (ZMRPALSH2, SEQ ID NO:4) using the GCG PileUp programutilizing default parameters. The putative zinc finger region is shownin italics.

[0024]FIG. 2 provides an expression construct for inducible expressionof the maize RPA large or middle subunit antisense construct.

DETAILED DESCRIPTION OF THE INVENTION

[0025] Nucleotide sequences and proteins useful for modulating DNAmetabolism are provided. The nucleotide and amino acid sequencescorrespond to the maize replication protein A (RPA) subunits. RPA is asingle-stranded DNA-binding protein that is required for multipleprocesses in DNA metabolism, including DNA replication, DNA repair, andrecombination. The RPA complex generally comprises subunits ofapproximately 70, 32, and 14 kDa. By “large subunit”, “middle subunit”,and “small subunit” is herein intended a RPA subunit having theapproximate molecular weight of 70-, 32-, and 14 kDa respectively. Thesequences of the invention comprise the large- and middle subunits ofthe RPA complex. The sequences of the invention additionally find use inmodulating gene expression.

[0026] Compositions of the invention include RPA nucleotide and aminoacid sequences that are involved in modulating DNA metabolism. Inparticular, the present invention provides for isolated nucleic acidmolecules comprising nucleotide sequences encoding the amino acidsequences shown in SEQ ID NOs: 2 and 4 for the large subunit, and SEQ IDNOs: 12, 14, 16, 18, 20, and 22 for the middle subunit. SEQ ID NO:2 andSEQ ID NO:4 correspond to the amino acid sequences for the maize RPAlarge subunit homologue 1 (ZmRPALSH1) and homologue 2 (ZmRPALSH2). SEQID NOs: 12, 14, 16, 18, 20, and 22 correspond to the amino acidsequences for the maize middle subunit homologue 1 (ZmRPAMSH1);homologues 2 and 3 (ZmRPAMSH2 and ZmRPAMSH3); homologue 4 (ZmRPAMSH4);homologue 5 (ZmRPAMSH5); homologue 6 (ZmRPAMSH6); and homologue 7(ZmRPAMSH7) respectively.

[0027] For the large subunit, the present invention alternativelyprovides the nucleotide sequences encoding the DNA sequences depositedin a bacterial host as Patent Deposit Nos: 98754 and 98843. For thelarge subunits, further are polypeptides having an amino acid sequenceencoded by a nucleic acid molecule described herein, for example thoseset forth in SEQ ID NOs: 1 and 3, those deposited in a bacterial host asPatent Deposit Nos: 98754 and 98843, and fragments and variants thereof.

[0028] Plasmids containing the RPA large subunit nucleotide sequences ofthe invention were deposited with the Patent Depository of the AmericanType Culture Collection (ATCC), Manassas, Va., and assigned PatentDeposit NOs: 98754 and 98843. These deposits will be maintained underthe terms of the Budapest Treaty on the International Recognition of theDeposit of Microorganisms for the Purposes of Patent Procedure. Thesedeposits were made merely as a convenience for those of skill in the artand are not an admission that a deposit is required under 35 U.S.C.§112.

[0029] Nucleotide sequences encoding the amino acid sequences for themaize RPA large subunit homologue 1 (ZmRPALSH1) and homologue 2(ZmRPALSH2) are set forth in SEQ ID NOs 1 and 3. Nucleotide sequencesencoding the amino acid sequences for the maize RPA middle subunithomologue 1 (ZmRPAMSH1); homologues 2 and 3 (ZmRPAMSH2 and ZmRPAMSH3);homologue 4 (ZmRPAMSH4); homologue 5 (ZmRPAMSH5); homologue 6(ZmRPAMSH6); and homologue 7 (ZmRPAMSH7) are set forth in SEQ ID NOs:11, 13, 15, 17, 19, and 21 respectively.

[0030] The invention encompasses isolated or substantially purifiednucleic acid or protein compositions. An “isolated” or “purified”nucleic acid molecule or protein, or biologically active portionthereof, is substantially free of other cellular material, or culturemedium when produced by recombinant techniques, or substantially free ofchemical precursors or other chemicals when chemically synthesized.Preferably, an “isolated” nucleic acid is free of sequences (preferablyprotein encoding sequences) that naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in thegenomic DNA of the organism from which the nucleic acid is derived. Forexample, in various embodiments, the isolated nucleic acid molecule cancontain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kbof nucleotide sequences that naturally flank the nucleic acid moleculein genomic DNA of the cell from which the nucleic acid is derived. Aprotein that is substantially free of cellular material includespreparations of protein having less than about 30%, 20%, 10%, 5%, (bydry weight) of contaminating protein. When the protein of the inventionor biologically active portion thereof is recombinantly produced,preferably culture medium represents less than about 30%, 20%, 10%, or5% (by dry weight) of chemical precursors or non-protein-of-interestchemicals.

[0031] RPA binds tightly to single-stranded DNA (ssDNA). The affinity ofbinding to double-stranded DNA (dsDNA) is three to four orders ofmagnitude lower than the binding affinity for ssDNA. Because RPA hasbeen found to bind specifically to certain dsDNA sequences that seem tobe involved in the regulation of transcription, modulation of geneexpression may be affected by an increase or decrease in RPA expressionin the host cell.

[0032] RPA has a wide range of activity and therefore uses relating toDNA metabolism and cell cycle. RPA interacts specifically with severalproteins required for nucleotide excision repair. Interactions withrepair proteins indicate that RPA may be important for efficient damagerecognition and cleavage. RPA additionally interacts with RAD52 protein,a protein that is essential for dsDNA-break repair. This interactionappears to be essential for homologous recombination. In this manner,expression of the nucleotides of the invention may promote homologousrecombination by recruiting factors which are essential forrecombination to occur. Thus, the methods and compositions of theinvention find use in promoting homologous recombination.

[0033] In one embodiment, genetic manipulation by homologousrecombination can be improved by either expression of the RPA codingsequences of the invention during transformation, or by providing RPAprotein. RPA protein, for example, may be provided as a coating toparticles during particle bombardment. Alternatively, DNA constructsproviding for the expression of RPA may be included with the DNA to betransformed. The increase in RPA during transformation, particularlyintegration of polynucleotides by homologous recombination, promotesintegration and insertion of the DNA sequences of interest into theplant genome.

[0034] In the same manner, it may be beneficial to inhibit theexpression or presence of the RPA protein to encourage non-specificrecombination events. In this manner, antibodies, peptides, antisenseoligonucleotides and the like may be utilized to inhibit the activity ofRPA. Alternatively, antisense constructs may be provided to inhibit theexpression of RPA and encourage non-specific recombination.

[0035] Catalytic RNA molecules or ribozymes can also be used to inhibitexpression of plant genes. It is possible to design ribozymes thatspecifically pair with virtually any target RNA and cleave thephosphodiester backbone at a specific location, thereby functionallyinactivating the target RNA. In carrying out this cleavage, the ribozymeis not itself altered, and is thus capable of recycling and cleavingother molecules, making it a true enzyme. The inclusion of ribozymesequences within antisense RNAs confers RNA-cleaving activity upon them,thereby increasing the activity of the constructs. The design and use oftarget RNA-specific ribozymes is described in Haseloff et al. (1988)Nature 334:585-591.

[0036] A variety of cross-linking agents, alkylating agents and radicalgenerating species as pendant groups on polynucleotides of the presentinvention can be used to bind, label, detect, and/or cleave nucleicacids. For example, Vlassov, V. V. et al. (1986) Nucleic Acids Res.14:4065-4076, describe covalent bonding of a single-stranded DNAfragment with alkylating derivatives of nucleotides complementary totarget sequences. A report of similar work by the same group is that byKnorre et al. (1985) Biochimie 67:785-789. Iverson and Dervan alsoshowed sequence-specific cleavage of single-stranded DNA mediated byincorporation of a modified nucleotide which was capable of activatingcleavage (1987) J. Am. Chem. Soc. 109:1241-1243). Meyer et al. (1989) J.Am. Chem. Soc. 111:8517-8519, effect covalent crosslinking to a targetnucleotide using an alkylating agent complementary to thesingle-stranded target nucleotide sequence. A photoactivatedcrosslinking to single-stranded oligonucleotides mediated by psoralenwas disclosed by Lee et al. (1988) Biochem. 27:3197-3203. Use ofcrosslinking in triple-helix forming probes was also disclosed by Homeet al. (1990) J. Am. Chem. Soc. 112:2435-2437. Use of N4,N4-ethanocytosine as an alkylating agent to crosslink to single-strandedoligonucleotides has also been described by Webb et al. (1986) J. Am.Chem. Soc. 108:2764-2765; Webb et al., (1986) Nucleic Acids Res.14:7661-7674; Feteritz et al. (1991) J. Am. Chem. Soc. 113:4000. Variouscompounds to bind, detect, label, and/or cleave nucleic acids are knownin the art. See, for example, U.S. Pat. Nos. 5,543,507; 5,672,593;5,484,908; 5,256,648; and 5,681,941.

[0037] RPA is required for the replication of chromosomal DNA.Inhibition of endogenous RPA expression is deleterious to the cell,organism, or plant. Thus, the constructs of the invention can be used toselectively kill target cells or tissues. This can be accomplishedthrough the use of inducible or tissue-preferred promoters. In thismanner, the sequences of the invention may find use in enhancingpathogen resistance. An antisense construct for the RPA coding sequenceis operably linked to a pathogen-inducible promoter. Upon contact withthe pathogen, the RPA antisense construct is expressed resulting in celldeath and effectively preventing the invasion of the pathogen.

[0038] The invention is drawn to compositions and methods for inducingresistance in a plant to plant pests. Accordingly, the compositions andmethods are also useful in protecting plants against fungal pathogens,viruses, nematodes, insects and the like.

[0039] By “disease resistance” is intended that the plants avoid thedisease symptoms that are the outcome of plant-pathogen interactions.That is, pathogens are prevented from causing plant diseases and theassociated disease symptoms, or alternatively, the disease symptomscaused by the pathogen is minimized or lessened. The methods of theinvention can be utilized to protect plants from disease, particularlythose diseases that are caused by plant pathogens.

[0040] Pathogens of the invention include, but are not limited to,viruses or viroids, bacteria, insects, nematodes, fungi, and the like.Viruses include any plant virus, for example, tobacco or cucumber mosaicvirus, ringspot virus, necrosis virus, maize dwarf mosaic virus, etc.Specific fungal and viral pathogens for the major crops include:Soybeans: Phytophthora megasperma fsp. glycinea, Macrophominaphaseolina, Rhizoctonia solani, Sclerotinia sclerotiorum, Fusariumoxysporum, Diaporthe phaseolorum var. sojae (Phomopsis sojae), Diaporthephaseolorum var. caulivora, Sclerotium rolfsii, Cercospora kikuchii,Cercospora sojina, Peronospora manshurica, Colletotrichum dematium(Colletotichum truncatum), Corynespora cassiicola, Septoria glycines,Phyllosticta sojicola, Alternaria alternata, Pseudomonas syringae p.v.glycinea, Xanthomonas campestris p.v. phaseoli, Microsphaera diffusa,Fusarium semitectum, Phialophora gregata, Soybean mosaic virus,Glomerella glycines, Tobacco Ring spot virus, Tobacco Streak virus,Phakopsora pachyrhizi, Pythium aphanidermatum, Pythium ultimum, Pythiumdebaryanum, Tomato spotted wilt virus, Heterodera glycines Fusariumsolani; Canola: Albugo candida, Alternaria brassicae, Leptosphaeriamaculans, Rhizoctonia solani, Sclerotinia sclerotiorum, Mycosphaerellabrassiccola, Pythium ultimum, Peronospora parasitica, Fusarium roseum,Alternaria alternata; Alfalfa: Clavibater michiganese subsp. insidiosum,Pythium ultimum, Pythium irregulare, Pythium splendens, Pythiumdebaryanum, Pythium aphanidermatum, Phytophthora megasperma, Peronosporatrifoliorum, Phoma medicaginis var. medicaginis, Cercospora medicaginis,Pseudopeziza medicaginis, Leptotrochila medicaginis, Fusarium,Xanthomonas campestris p.v. alfalfae, Aphanomyces euteiches, Stemphyliumherbarum, Stemphylium alfalfae; Wheat: Pseudomonas syringae p.v.atrofaciens, Urocystis agropyri, Xanthomonas campestris p.v.translucens, Pseudomonas syringae p.v. syringae, Alternaria alternata,Cladosporium herbarum, Fusarium graminearum, Fusarium avenaceum,Fusarium culmorum, Ustilago tritici, Ascochyta tritici, Cephalosporiumgramineum, Collotetrichum graminicola, Erysiphe graminis f.sp. tritici,Puccinia graminis f.sp. tritici, Puccinia recondite f.sp. tritici,Puccinia striiformis, Pyrenophora tritici-repentis, Septoria nodorum,Septoria tritici, Septoria avenae, Pseudocercosporella herpotrichoides,Rhizoctonia solani, Rhizoctonia cerealis, Gaeumannomyces graminis var.tritici, Pythium aphanidermatum, Pythium arrhenomanes, Pythium ultimum,Bipolaris sorokiniana, Barley Yellow Dwarf Virus, Brome Mosaic Virus,Soil Borne Wheat Mosaic Virus, Wheat Streak Mosaic Virus, Wheat SpindleStreak Virus, American Wheat Striate Virus, Claviceps purpurea, Tilletiatritici, Tilletia laevis, Ustilago tritici, Tilletia indica, Rhizoctoniasolani, Pythium arrhenomannes, Pythium gramicola, Pythiumaphanidermatum, High Plains Virus, European wheat striate virus;Sunflower: Plasmophora halstedii, Sclerotinia sclerotiorum, AsterYellows, Septoria helianthi, Phomopsis helianthi, Altemaria helianthi,Alternaria zinniae, Botrytis cinerea, Phoma macdonaldii, Macrophominaphaseolina, Erysiphe cichoracearum, Rhizopus oryzae, Rhizopus arrhizus,Rhizopus stolonifer, Puccinia helianthi, Verticillium dahliae, Erwiniacarotovorum pv. carotovora, Cephalosporium acremonium, Phytophthoracryptogea, Albugo tragopogonis; Corn: Fusarium moniliforme var.subglutinans, Erwinia stewartii, Fusarium moniliforme, Gibberella zeae(Fusarium graminearum), Stenocarpella maydi (Diplodia maydis), Pythiumirregulare, Pythium debaryanum, Pythium graminicola, Pythium splendens,Pythium ultimum, Pythium aphanidermatum, Aspergillus flavus, Bipolarismaydis O, T (Cochliobolus heterostrophus), Helminthosporium carbonum I,II & III (Cochliobolus carbonum), Exserohilum turcicum I, II & III,Helminthosporium pedicellatum, Physoderma maydis, Phyllosticta maydis,Kabatiella maydis, Cercospora sorghi, Ustilago maydis, Puccinia sorghi,Puccinia polysora, Macrophomina phaseolina, Penicillium oxalicum,Nigrospora oryzae, Cladosporium herbarum, Curvularia lunata, Curvulariainaequalis, Curvularia pallescens, Clavibacter michiganense subsp.nebraskense, Trichoderma viride, Maize Dwarf Mosaic Virus A & B, WheatStreak Mosaic Virus, Maize Chlorotic Dwarf Virus, Claviceps sorghi,Pseudonomas avenae, Erwinia chrysanthemi pv. zea, Erwinia carotovora,Corn stunt spiroplasma, Diplodia macrospora, Sclerophthora macrospora,Peronosclerospora sorghi, Peronosclerospora philippinensis,Peronosclerospora maydis, Peronosclerospora sacchari, Sphacelothecareiliana, Physopella zeae, Cephalosporium maydis, Cephalosporiumacremonium, Maize Chlorotic Mottle Virus, High Plains Virus, MaizeMosaic Virus, Maize Rayado Fino Virus, Maize Streak Virus, Maize StripeVirus, Maize Rough Dwarf Virus; Sorghum: Exserohilum turcicum,Colletotrichum graminicola (Glomerella graminicola), Cercospora sorghi,Gloeocercospora sorghi, Ascochyta sorghina, Pseudomonas syringae p.v.syringae, Xanthomonas campestris p.v. holcicola, Pseudomonasandropogonis, Puccinia purpurea, Macrophomina phaseolina, Perconiacircinata, Fusarium moniliforme, Alternaria alternata, Bipolarissorghicola, Helminthosporium sorghicola, Curvularia lunata, Phomainsidiosa, Pseudomonas avenae (Pseudomonas alboprecipitans), Ramulisporasorghi, Ramulispora sorghicola, Phyllachara sacchari, Sporisoriumreilianum (Sphacelotheca reiliana), Sphacelotheca cruenta, Sporisoriumsorghi, Sugarcane mosaic H, Maize Dwarf Mosaic Virus A & B, Clavicepssorghi, Rhizoctonia solani, Acremonium strictum, Sclerophthonamacrospora, Peronosclerospora sorghi, Peronosclerospora philippinensis,Sclerospora graminicola, Fusarium graminearum, Fusarium oxysporum,Pythium arrhenomanes, Pythium graminicola, etc.

[0041] Nematodes include parasitic nematodes such as root-knot, cyst,and lesion nematodes, including Heterodera and Globodera spp;particularly Globodera rostochiensis and globodera pailida (potato cystnematodes); Heterodera glycines (soybean cyst nematode); Heteroderaschachtii (beet cyst nematode); and Heterodera avenae (cereal cystnematode).

[0042] Insect pests include insects selected from the orders Coleoptera,Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera,Orthoptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera,Trichoptera, etc., particularly Coleoptera and Lepidoptera. Insect pestsof the invention for the major crops include: Maize: Ostrinia nubilalis,European corn borer; Agrotis ipsilon, black cutworm; Helicoverpa zea,corn earworm; Spodoptera frugiperda, fall armyworm; Diatraeagrandiosella, southwestern corn borer; Elasmopalpus lignosellus, lessercornstalk borer; Diatraea saccharalis, surgarcane borer; Diabroticavirgifera, western corn rootworm; Diabrotica longicornis barberi,northern corn rootworm; Diabrotica undecimpunctata howardi, southerncorn rootworm; Melanotus spp., wireworms; Cyclocephala borealis,northern masked chafer (white grub); Cyclocephala immaculata, southernmasked chafer (white grub); Popillia japonica, Japanese beetle;Chaetocnema pulicaria, corn flea beetle; Sphenophorus maidis, maizebillbug; Rhopalosiphum maidis, corn leaf aphid; Anuraphis maidiradicis,corn root aphid; Blissus leucopterus leucopterus, chinch bug; Melanoplusfemurrubrum, redlegged grasshopper; Melanoplus sanguinipes, migratorygrasshopper; Hylemya platura, seedcorn maggot; Agromyza parvicornis,corn blot leafminer; Anaphothrips obscrurus, grass thrips; Solenopsismilesta, thief ant; Tetranychus urticae, twospotted spider mite;Sorghum: Chilo partellus, sorghum borer; Spodoptera frugiperda, fallarmyworm; Helicoverpa zea, corn earworm; Elasmopalpus lignosellus,lesser cornstalk borer; Feltia subterranea, granulate cutworm;Phyllophaga crinita, white grub; Eleodes, Conoderus, and Aeolus spp.,wireworms; Oulema melanopus, cereal leaf beetle; Chaetocnema pulicaria,corn flea beetle; Sphenophorus maidis, maize billbug; Rhopalosiphummaidis; corn leaf aphid; Sipha flava, yellow sugarcane aphid; Blissusleucopterus leucopterus, chinch bug; Contarinia sorghicola, sorghummidge; Tetranychus cinnabarinus, carmine spider mite; Tetranychusurticae, twospotted spider mite; Wheat: Pseudaletia unipunctata, armyworm; Spodoptera frugiperda, fall armyworm; Elasmopalpus lignosellus,lesser cornstalk borer; Agrotis orthogonia, western cutworm;Elasmopalpus lignosellus, lesser cornstalk borer; Oulema melanopus,cereal leaf beetle; Hypera punctata, clover leaf weevil; Diabroticaundecimpunctata howardi, southern corn rootworm; Russian wheat aphid;Schizaphis graminum, greenbug; Macrosiphum avenae, English grain aphid;Melanoplus femurrubrum, redlegged grasshopper; Melanoplusdifferentialis, differential grasshopper; Melanoplus sanguinipes,migratory grasshopper; Mayetiola destructor, Hessian fly; Sitodiplosismosellana, wheat midge; Meromyza americana, wheat stem maggot; Hylemyacoarctata, wheat bulb fly; Frankliniella fusca, tobacco thrips; Cephuscinctus, wheat stem sawfly; Aceria tulipae, wheat curl mite; Sunflower:Suleima helianthana, sunflower bud moth; Homoeosoma electellum,sunflower moth; zygogramma exclamationis, sunflower beetle; Bothyrusgibbosus, carrot beetle; Neolasioptera murtfeldtiana, sunflower seedmidge; Cotton: Heliothis virescens, cotton budworm; Helicoverpa zea,cotton bollworm; Spodoptera exigua, beet armyworm; Pectinophoragossypiella, pink bollworm; Anthonomus grandis grandis, boll weevil;Aphis gossypii, cotton aphid; Pseudatomoscelis seriatus, cottonfleahopper; Trialeurodes abutilonea, bandedwinged whitefly; Lyguslineolaris, tarnished plant bug; Melanoplus femurrubrum, redleggedgrasshopper; Melanoplus differentialis, differential grasshopper; Thripstabaci, onion thrips; Franklinkiella fusca, tobacco thrips; Tetranychuscinnabarinus, carmine spider mite; Tetranychus urticae, twospottedspider mite; Rice: Diatraea saccharalis, sugarcane borer; Spodopterafrugiperda, fall armyworm; Helicoverpa zea, corn earworm; Colaspisbrunnea, grape colaspis; Lissorhoptrus oryzophilus, rice water weevil;Sitophilus oryzae, rice weevil; Nephotettix nigropictus, riceleafhopper; Blissus leucopterus leucopterus, chinch bug; Acrosternumhilare, green stink bug; Soybean: Pseudoplusia includens, soybeanlooper; Anticarsia gemmatalis, velvetbean caterpillar; Plathypenascabra, green cloverworm; Ostrinia nubilalis, European corn borer;Agrotis ipsilon, black cutworm; Spodoptera exigua, beet armyworm;Heliothis virescens, cotton budworm; Helicoverpa zea, cotton bollworm;Epilachna varivestis, Mexican bean beetle; Myzus persicae, green peachaphid; Empoasca fabae, potato leafhopper; Acrosternum hilare, greenstink bug; Melanoplus femurrubrum, redlegged grasshopper; Melanoplusdifferentialis, differential grasshopper; Hylemya platura, seedcornmaggot; Sericothrips variabilis, soybean thrips; Thrips tabaci, onionthrips; Tetranychus turkestani, strawberry spider mite; Tetranychusurticae, twospotted spider mite; Barley: Ostrinia nubilalis, Europeancorn borer; Agrotis ipsilon, black cutworm; Schizaphis graminum,greenbug; Blissus leucopterus leucopterus, chinch bug; Acrostemumhilare, green stink bug; Euschistus servus, brown stink bug; Deliaplatura, seedcorn maggot; Mayetiola destructor, Hessian fly; Petrobialatens, brown wheat mite; Oil Seed Rape: Brevicoryne brassicae, cabbageaphid; Phyllotreta cruciferae, Flea beetle; Mamestra configurata, Berthaarmyworm; Plutella xylostella, Diamond-back moth; Delia ssp., Rootmaggots.

[0043] A number of promoters can be used in the practice of theinvention. The promoters can be selected based on the desired outcome.The nucleic acids can be combined with constitutive, tissue-preferred,or other promoters for expression in plants.

[0044] A plant promoter can be employed which will direct expression ofa polynucleotide of the present invention in all tissues of aregenerated plant. Such promoters are referred to herein as“constitutive” promoters and are active under most environmentalconditions and states of development or cell differentiation. Suchconstitutive promoters include, for example, the core promoter of theRsyn7 (WO 99/43838); the core CaMV 35S promoter (Odell et al. (1985)Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol.12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689);pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten etal. (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026),and the like. Other constitutive promoters include, for example, U.S.Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785;5,399,680; 5,268,463; and 5,608,142.

[0045] Alternatively, the plant promoter can direct expression of apolynucleotide of present invention in a specific tissue or may beotherwise under more precise environmental or developmental control.Such promoters are referred to here as “inducible” promoters.Environmental conditions that may effect transcription by induciblepromoters include pathogen attack, anaerobic conditions, or the presenceof light. Examples of inducible promoters are the Adhl promoter which isinducible by hypoxia or cold stress, the Hsp70 promoter which isinducible by heat stress, and the PPDK promoter which is inducible bylight.

[0046] Examples of promoters under developmental control includepromoters that initiate transcription only, or preferentially, incertain tissues, such as leaves, roots, fruit, seeds, or flowers. Anexemplary promoter is the anther specific promoter 5126 (U.S. Pat. Nos.5,689,049 and 5,689,051). The operation of a promoter may also varydepending on its location in the genome. Thus, an inducible promoter maybecome fully or partially constitutive in certain locations.

[0047] The promoters can be selected based on the desired outcome. Whenthe genes are expressed at levels to cause cell death, an induciblepromoter or tissue specific promoters can be used to drive theexpression of the genes of the invention. The inducible promoter must betightly regulated to prevent unnecessary cell death, yet be expressed inthe presence of a pathogen to prevent infection and disease symptoms.

[0048] Generally, it will be beneficial to express the gene from aninducible promoter, particularly from a pathogen-inducible promoter.Such promoters include those from pathogenesis-related proteins (PRproteins), which are induced following infection by a pathogen; e.g., PRproteins, SAR proteins, beta-1,3-glucanase, chitinase, etc. See, forexample, Redolfi et al. (1983) Neth. J. Plant Pathol. 89:245-254; Ukneset al. (1992) Plant Cell 4:645-656; and Van Loon (1985) Plant Mol.Virol. 4:111-116. See also U.S. Pat. No. 6,429,362, herein incorporatedby reference.

[0049] Of interest are promoters that are expressed locally at or nearthe site of pathogen infection. See, for example, Marineau et al. (1987)Plant Mol. Biol. 9:335-342; Matton et al. (1989) Molecular Plant-MicrobeInteractions 2:325-331; Somsisch et al. (1986) Proc. Natl. Acad. Sci.USA 83:2427-2430; Somsisch et al. (1988) Mol. Gen. Genet. 2:93-98; andYang (1996) Proc. Natl. Acad. Sci. USA 93:14972-14977. See also, Chen etal. (1996) Plant J. 10:955-966; Zhang et al. (1994) Proc. Natl. Acad.Sci. USA 91:2507-2511; Warner et al. (1993) Plant J. 3:191-201; Siebertzet al. (1989) Plant Cell 1:961-968; U.S. Pat. No. 5,750,386(nematode-inducible); and the references cited therein. Of particularinterest is the inducible promoter for the maize PRms gene, whoseexpression is induced by the pathogen Fusarium moniliforme (see, forexample, Cordero et al. (1992) Physiol. Mol. Plant Path. 41:189-200).

[0050] Additionally, as pathogens find entry into plants through woundsor insect damage, a wound-inducible promoter may be used in theconstructions of the invention. Such wound-inducible promoters includepotato proteinase inhibitor (pin II) gene (Ryan (1990) Ann. Rev.Phytopath. 28:425-449; Duan et al. (1996) Nature Biotechnology14:494498); wun1 and wun2, U.S. Pat. No. 5,428,148; win1 and win2(Stanford et al. (1989) Mol. Gen. Genet. 215:200-208); systemin (McGurlet al. (1992) Science 225:1570-1573); WIP1 (Rohmeier et al. (1993) PlantMol. Biol. 22:783-792; Eckelkamp et al. (1993) FEBS Letters 323:73-76);MPI gene (Corderok et al. (1994) Plant J. 6(2):141-150); and the like,herein incorporated by reference.

[0051] Chemical-regulated promoters can be used to modulate theexpression of a gene in a plant through the application of an exogenouschemical regulator. Depending upon the objective, the promoter may be achemical-inducible promoter, where application of the chemical inducesgene expression, or a chemical-repressible promoter, where applicationof the chemical represses gene expression. Chemical-inducible promotersare known in the art and include, but are not limited to, the maizeIn2-2 promoter, which is activated by benzenesulfonamide herbicidesafeners, the maize GST promoter, which is activated by hydrophobicelectrophilic compounds that are used as pre-emergent herbicides, andthe tobacco PR-1a promoter, which is activated by salicylic acid. Otherchemical-regulated promoters of interest include steroid-responsivepromoters (see, for example, the glucocorticoid-inducible promoter inSchena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 andMcNellis et al., (1998) Plant J. 14(2):247-257) andtetracycline-inducible and tetracycline-repressible promoters (see, forexample, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and U.S. Pat.Nos. 5,814,618 and 5,789,156), herein incorporated by reference.

[0052] Where low level expression is desired, weak promoters will beused. Generally, by “weak promoter” is intended a promoter that drivesexpression of a coding sequence at a low level. By low level is intendedat levels of about {fraction (1/1000)} transcripts to about {fraction(1/100,000)} transcripts to about {fraction (1/500,000)} transcripts.Alternatively, it is recognized that weak promoters also encompassespromoters that are expressed in only a few cells and not in others togive a total low level of expression. Where a promoter is expressed atunacceptably high levels, portions of the promoter sequence can bedeleted or modified to decrease expression levels.

[0053] Such weak constitutive promoters include, for example, the corepromoter of the Rsyn7 (WO 99/43838), the core 35S CaMV promoter, and thelike. Other constitutive promoters include, for example, U.S. Pat. Nos.5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680;5,268,463; and 5,608,142. See also, U.S. Pat. No. 6,177,611, hereinincorporated by reference.

[0054] Tissue-preferred promoters can be utilized to target enhanced RPAexpression within a particular plant tissue. In this aspect of theinvention, the antisense constructs are useful for tissue-preferredexpression. Male or female sterility may be affected by use of theantisense constructs with tissue-preferred promoters. Although not alimitation, of particular interest are promoters for male sterility. Forexample, the anther-preferred promoter 5126 can be used. See, forexample, U.S. Pat. Nos. 5,689,049 and 5,689,051, herein incorporated byreference.

[0055] Tissue-preferred promoters include Yamamoto et al. (1997) PlantJ. 12(2)255-265; Kawamata et al. (1997) Plant Cell Physiol.38(7):792-803; Hansen et al. (1997) Mol. Gen Genet. 254(3):337-343;Russell et al. (1997) Transgenic Res. 6(2):157-168; Rinehart et al.(1996) Plant Physiol. 112(3):1331-1341; Van Camp et al. (1996) PlantPhysiol. 112(2):525-535; Canevascini et al. (1996) Plant Physiol.112(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol.35(5):773-778; Lam (1994) Results Probl. Cell Differ. 20:181-196; Orozcoet al. (1993) Plant Mol Biol. 23(6): 1129-1138; Matsuoka et al. (1993)Proc Natl. Acad. Sci. USA 90(20):9586-9590; and Guevara-Garcia et al.(1993) Plant J. 4(3):495-505. Such promoters can be modified, ifnecessary, for weak expression.

[0056] Leaf-specific promoters are known in the art. See, for example,Yamamoto et al. (1997) Plant J. 12(2):255-265; Kwon et al. (1994) PlantPhysiol. 105:357-67; Yamamoto et al. (1994) Plant Cell Physiol.35(5):773-778; Gotor et al. (1993) Plant J. 3:509-18; Orozco et al.(1993) Plant Mol. Biol. 23(6):1129-1138; and Matsuoka et al. (1993)Proc. Natl. Acad. Sci. USA 90(20):9586-9590.

[0057] Root-specific promoters are known and can be selected from themany available from the literature or isolated de novo from variouscompatible species. See, for example, Hire et al. (1992) Plant Mol.Biol. 20(2): 207-218 (soybean root-specific glutamine synthetase gene);Keller and Baumgartner (1991) Plant Cell 3(10):1051-1061 (root-specificcontrol element in the GRP 1.8 gene of French bean); Sanger et al.(1990) Plant Mol. Biol. 14(3):433-443 (root-specific promoter of themannopine synthase (MAS) gene of Agrobacterium tumefaciens); and Miao etal. (1991) Plant Cell 3(1):11-22 (full-length cDNA clone encodingcytosolic glutamine synthetase (GS), which is expressed in roots androot nodules of soybean). See also Bogusz et al. (1990) Plant Cell2(7):633-641, where two root-specific promoters isolated from hemoglobingenes from the nitrogen-fixing nonlegume Parasponia andersonii and therelated non-nitrogen-fixing nonlegume Trema tomentosa are described. Thepromoters of these genes were linked to a β-glucuronidase reporter geneand introduced into both the nonlegume Nicotiana tabacum and the legumeLotus corniculatus, and in both instances root-specific promoteractivity was preserved. Leach and Aoyagi (1991) describe their analysisof the promoters of the highly expressed roIC and roID root-inducinggenes of Agrobacterium rhizogenes (see Plant Science (Limerick)79(1):69-76). They concluded that enhancer and tissue-preferred DNAdeterminants are dissociated in those promoters. Teeri et al. (1989)used gene fusion to lacZ to show that the Agrobacterium T-DNA geneencoding octopine synthase is especially active in the epidermis of theroot tip and that the TR2′ gene is root specific in the intact plant andstimulated by wounding in leaf tissue, an especially desirablecombination of characteristics for use with an insecticidal orlarvicidal gene (see EMBO J. 8(2):343-350). The TR1′ gene, fused tonptll (neomycin phosphotransferase II) showed similar characteristics.Additional root-preferred promoters include the VfENOD-GRP3 genepromoter (Kuster et al. (1995) Plant Mol. Biol. 29(4):759-772); and rolBpromoter (Capana et al., (1994) Plant Mol. Biol. 25(4):681-691. See alsoU.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836;5,110,732; and 5,023,179.

[0058] “Seed-preferred” promoters include both “seed-specific” promoters(those promoters active during seed development such as promoters ofseed storage proteins) as well as “seed-germinating” promoters (thosepromoters active during seed germination). See Thompson et al. (1989)BioEssays 10:108, herein incorporated by reference. Such seed-preferredpromoters include, but are not limited to, Cim1 (cytokinin-inducedmessage); cZ19B1 (maize 19 kDa zein); milps (myo-inositol-1-phosphatesynthase); and celA (cellulose synthase) (see U.S. Pat. No. 6,225,529,herein incorporated by reference). Gamma-zein is a preferredendosperm-specific promoter. Glob-1 is a preferred embryo-specificpromoter. For dicots, seed-specific promoters include, but are notlimited to, bean β-phaseolin, napin, β-conglycinin, soybean lectin,cruciferin, and the like. For monocots, seed-specific promoters include,but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa zein,g-zein, waxy, shrunken 1, shrunken 2, globulin 1, etc.

[0059] Both heterologous and non-heterologous (i.e., endogenous)promoters can be employed to direct expression of the nucleic acids ofthe present invention. These promoters can also be used, for example, inrecombinant expression cassettes to drive expression of antisensenucleic acids to reduce, increase, or alter RPA content and/orcomposition in a desired tissue, or to generate sterile plants.Optionally, RPA nucleic acids from a variety of sources, as discussedabove can be employed to create male sterile plants. In optionalembodiments, the RPA gene or cDNA is operably linked to ananther-specific promoter such as 5126, as discussed above. Preferably,the male sterile plant is maize.

[0060] Thus, in some embodiments, the nucleic acid construct willcomprise a promoter functional in a plant cell, such as in Zea mays,operably linked to a polynucleotide of the present invention. Promotersuseful in these embodiments include the endogenous promoters drivingexpression of a polypeptide of the present invention.

[0061] In some embodiments, isolated nucleic acids which serve aspromoter or enhancer elements can be introduced in the appropriateposition (generally upstream) of a non-heterologous form of apolynucleotide of the present invention so as to up or down regulateexpression of a polynucleotide of the present invention. For example,endogenous promoters can be altered in vivo by mutation, deletion,and/or substitution (see, Kmiec, U.S. Pat. No. 5,565,350; Zarling etal., PCT/US93/03868), or isolated promoters can be introduced into aplant cell in the proper orientation and distance from a RPA gene so asto control the expression of the gene. Gene expression can be modulatedunder conditions suitable for plant growth so as to alter RPA contentand/or composition. Thus, the present invention provides compositions,and methods for making, heterologous promoters and/or enhancers operablylinked to a native, endogenous (i.e., non-heterologous) form of apolynucleotide of the present invention.

[0062] Methods for identifying promoters with a particular expressionpattern, in terms of e.g., tissue type, cell type, stage of development,and/or environmental conditions, are well known in the art. See, e.g.,The Maize Handbook, Chapters 114-115, Freeling and Walbot, eds.,Springer, N.Y. (1994); Corn and Corn Improvement, 3^(rd) edition,Chapter 6, Sprague and Dudley, eds., American Society of Agronomy,Madison, Wis. (1988). A typical step in promoter isolation methods isidentification of gene products that are expressed with some degree ofspecificity in the target tissue. Amongst the range of methodologiesare: differential hybridization to cDNA libraries; subtractivehybridization; differential display; differential 2-D protein gelelectrophoresis; DNA probe arrays; and isolation of proteins known to beexpressed with some specificity in the target tissue. Such methods arewell known to those of skill in the art. Commercially available productsfor identifying promoters are known in the art such as Clontech's (PaloAlto, Calif.) Universal GenomeWalker Kit.

[0063] For the protein-based methods, it is helpful to obtain the aminoacid sequence for at least a portion of the identified protein, and thento use the protein sequence as the basis for preparing a nucleic acidthat can be used as a probe to identify either genomic DNA directly, orpreferably, to identify a cDNA clone from a library prepared from thetarget tissue. Once such a cDNA clone has been identified, that sequencecan be used to identify the sequence at the 5′ end of the transcript ofthe indicated gene. For differential hybridization, subtractivehybridization and differential display, the nucleic acid sequenceidentified as enriched in the target tissue is used to identify thesequence at the 5′ end of the transcript of the indicated gene. Oncesuch sequences are identified, starting either from protein sequences ornucleic acid sequences, any of these sequences identified as being fromthe gene transcript can be used to screen a genomic library preparedfrom the target organism. Methods for identifying and confirming thetranscriptional start site are well known in the art.

[0064] In the process of isolating promoters expressed under particularenvironmental conditions or stresses, or in specific tissues, or atparticular developmental stages, a number of genes are identified thatare expressed under the desired circumstances, in the desired tissue, orat the desired stage. Further analysis will reveal expression of eachparticular gene in one or more other tissues of the plant. One canidentify a promoter with activity in the desired tissue or condition butthat do not have activity in any other common tissue.

[0065] To identify the promoter sequence, the 5′ portions of the clonesdescribed here are analyzed for sequences characteristic of promotersequences. For instance, promoter sequence elements include the TATA boxconsensus sequence (TATAAT), which is usually an AT-rich stretch of 5-10bp located approximately 20 to 40 base pairs upstream of thetranscription start site. Identification of the TATA box is well knownin the art. For example, one way to predict the location of this elementis to identify the transcription start site using standard RNA-mappingtechniques such as primer extension, S1 analysis, and/or RNaseprotection. To confirm the presence of the AT-rich sequence, astructure-function analysis can be performed involving mutagenesis ofthe putative region and quantification of the mutation's effect onexpression of a linked downstream reporter gene. See, e.g., The MaizeHandbook, Chapter 114, Freeling and Walbot, eds., Springer, N.Y. (1994).

[0066] In plants, further upstream from the TATA box, at positions −80to −100, there is typically a promoter element (i.e., the CAAT box) witha series of adenines surrounding the trinucleotide G (or T) N G. J.Messing et al., in Genetic Engineering in Plants, Kosage, Meredith andHollaender, eds., pp. 221-227 (1983). In maize, there no well-conservedCAAT box but there are several short, conserved protein-binding motifsupstream of the TATA box. These include motifs for the transactingtranscription factors involved in light regulation, anaerobic induction,hormonal regulation, or anthocyanin biosynthesis, as appropriate foreach gene.

[0067] Once promoter and/or gene sequences are known, a region ofsuitable size is selected from the genomic DNA that is 5′ to thetranscriptional start, or the translational start site, and suchsequences are then linked to a coding sequence. If the transcriptionalstart site is used as the point of fusion, any of a number of possible5′ untranslated regions can be used in between the transcriptional startsite and the partial coding sequence. If the translational start site atthe 3′ end of the specific promoter is used, then it is linked directlyto the methionine start codon of a coding sequence.

[0068] If polypeptide expression is desired, it is generally desirableto include a polyadenylation region at the 3′-end of a polynucleotidecoding region. The polyadenylation region can be derived from thenatural gene, from a variety of other plant genes, or from T-DNA. The 3′end sequence to be added can be derived from, example, the nopalinesynthase or octopine synthase genes, or alternatively from another plantgene, or less preferably from any other eukaryotic gene.

[0069] An intron sequence can be added to the 5′ untranslated region orthe coding sequence of the partial coding sequence to increase theamount of the mature message that accumulates in the cytosol. Inclusionof a spliceable intron in the transcription unit in both plant andanimal expression constructs has been shown to increase gene expressionat both the mRNA and protein levels up to 1000-fold. Buchman et al.(1988) Mol. Cell Biol. 8:4395-4405; Callis et al. (1987) Genes Dev.1:1183-1200. Such intron enhancement of gene expression is typicallygreatest when placed near the 5′ end of the transcription unit. Use ofmaize introns Adhl-S intron 1, 2, and 6, the Bronze-I intron are knownin the art. See generally, The Maize Handbook, Chapter 116, Freeling andWalbot, eds., Springer, N.Y. (1994).

[0070] The vector comprising the sequences from a polynucleotide of thepresent invention could comprise a selectable marker gene for theselection of transformed cells or tissues. Selectable marker genesinclude genes encoding antibiotic resistance, such as those encodingneomycin phosphotransferase II (NEO) and hygromycin phosphotransferase(HPT), as well as genes conferring resistance to herbicidal compounds,such as glufosinate ammonium, bromoxynil, imidazolinones, and2,4-dichlorophenoxyacetate (2,4-D). See generally, Yarranton (1992)Curr. Opin. Biotech. 3:506-511; Christopherson et al. (1992) Proc. Natl.Acad. Sci. USA 89:6314-6318; Yao et al. (1992) Cell 71:63-72; Reznikoff(1992) Mol. Microbiol. 6:2419-2422; Barkley et al. (1980) in The Operon,pp. 177-220; Hu et al. (1987) Cell 48:555-566; Brown et al. (1987) Cell49:603-612; Figge et al. (1988) Cell 52:713-722; Deuschle et al. (1989)Proc. Natl. Acad. Aci. USA 86:5400-5404; Fuerst et al. (1989) Proc.Natl. Acad. Sci. USA 86:2549-2553; Deuschle et al. (1990) Science248:480483; Gossen (1993) Ph.D. Thesis, University of Heidelberg; Reineset a. (1993) Proc. Natl. Acad. Sci. USA 90:1917-1921; Labow et al.(1990) Mol. Cell. Biol. 10:3343-3356; Zambretti et al., (1992) Proc.Natl. Acad. Sci. USA 89:3952-3956; Baim eta!. (1991) Proc. Natl. Acad.Sci. USA 88:5072-5076; Wyborski et a. (1991) Nucleic Acids Res.19:46474653; Hillenand-Wissman (1989) Topics Mol. Struc. Biol.10:143-162; Degenkolb eta!. (1991) Antimicrob. Agents Chemother.35:1591-1595; Kleinschmidt et al. (1988) Biochemistry 27:1094-1104;Bonin (1993) Ph.D. Thesis, University of Heidelberg; Gossen et a. (1992)Proc. Natl. Acad. Sci. USA 89:5547-5551; Oliva et al., (1992)Antimicrob. Agents Chemother. 36:913-919; Hlavka et al. (1985) Handbookof Experimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin); Gill etal. (1988) Nature 334:721-724. Such disclosures are herein incorporatedby reference.

[0071] The above list of selectable marker genes is not meant to belimiting. Any selectable marker gene can be used in the presentinvention.

[0072] Typical vectors useful for expression of genes in higher plantsare well known in the art and include vectors derived from thetumor-inducing (Ti) plasmid of Agrobacterium tumefaciens described byRogers et al. (1987) Meth. in Enzymol. 153:253-277. These vectors areplant integrating vectors in that on transformation, the vectorsintegrate a portion of vector DNA into the genome of the host plant.Exemplary A. tumefaciens vectors useful herein are plasmids pKYLX6 andpKYLX7 of Schardl et al. (1987) Gene 61:1-11 and Berger et al. (1989)Proc. Natl. Acad. Sci. (USA) 86:8402-8406. Another useful vector hereinis plasmid pBI101.2 that is available from Clontech Laboratories, Inc.(Palo Alto, Calif.).

[0073] As discussed above, a polynucleotide of the present invention canbe expressed in either sense or antisense orientation as desired. Itwill be appreciated that control of gene expression in either sense orantisense orientation can have a direct impact on the observable plantcharacteristics. Antisense technology can be conveniently used for geneexpression in plants. To accomplish this, a nucleic acid segment fromthe desired gene is cloned and operably linked to a promoter such thatthe antisense strand of RNA will be transcribed. The construct is thentransformed into plants and the antisense strand of RNA is produced. Inplant cells, it has been shown that antisense RNA inhibits geneexpression by preventing the accumulation of mRNA which encodes theenzyme of interest, see, e.g., Sheehy et al. (1988) Proc. Natl. Acad.Sci. (USA) 85:8805-8809; and Hiatt et al., U.S. Pat. No. 4,801,340.

[0074] In the methods of the invention, it is recognized that the entirecoding sequence for the RPA construct may be utilized. Alternatively,portions or fragments of the sequence may be used in DNA constructs.

[0075] Fragments and variants of the disclosed nucleotide sequences andproteins encoded thereby are encompassed by the present invention. By“fragment” is intended a portion of the nucleotide sequence or a portionof the amino acid sequence and hence protein encoded thereby. Fragmentsof a nucleotide sequence may encode protein fragments that retain thebiological activity of the native protein and hence modulate DNAmetabolism. Alternatively, fragments of a nucleotide sequence that areuseful as hybridization probes generally do not encode fragment proteinsretaining biological activity. Thus, fragments of a nucleotide sequencemay range from at least about 20 nucleotides, about 50 nucleotides,about 100 nucleotides, and up to the full-length nucleotide sequenceencoding the proteins of the invention.

[0076] A fragment of a RPA nucleotide sequence that encodes abiologically active portion of a RPA protein of the invention willencode at least 15, 25, 30, 50, 100, 150, 200, or 250 contiguous aminoacids, or up to the total number of amino acids present in a full-lengthRPA protein of the invention (for example, 623, 617, 273, 273, 273, 318,273, 273 amino acids for SEQ ID NOs: 2, 4, 12, 14, 16, 18, 20, and 22respectively. Fragments of a RPA nucleotide sequence that are useful ashybridization probes for PCR primers generally need not encode abiologically active portion of a RPA protein.

[0077] Thus, a fragment of a RPA nucleotide sequence may encode abiologically active portion of a RPA protein, or it may be a fragmentthat can be used as a hybridization probe or PCR primer using methodsdisclosed below. A biologically active portion of a RPA protein can beprepared by isolating a portion of one of the RPA nucleotide sequencesof the invention, expressing the encoded portion of the RPA protein(e.g., by recombinant expression in vitro), and assessing the activityof the encoded portion of the RPA protein. Nucleic acid molecules thatare fragments of a RPA nucleotide sequence comprise at least 16, 20, 50,75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,800, 900, 1,000 nucleotides, or up to the number of nucleotides presentin a full-length RPA nucleotide sequence disclosed herein (for example,2497, 2202, 1124, 979, 1051, 1087, 1074, and 1231 nucleotides for SEQ IDNOs: 1, 3, 11, 13, 15, 17, 19, and 21 respectively.

[0078] By “variants” is intended substantially similar sequences. Fornucleotide sequences, conservative variants include those sequencesthat, because of the degeneracy of the genetic code, encode the aminoacid sequence of one of the RPA polypeptides of the invention. Suchnaturally occurring variants including naturally occurring allelicvariants, can be identified with the use of well-known molecular biologytechniques, as, for example, with polymerase chain reaction (PCR) andhybridization techniques as outlined below. Variant nucleotide sequencesalso include synthetically derived nucleotide sequences, such as thosegenerated, for example, by using site-directed mutagenesis but whichstill encode a RPA protein of the invention. Generally, variants of aparticular nucleotide sequence of the invention will have at least 40%,50%, 60%, 70%, generally at least 75%, 80%, 85%, preferably about 90% to95% or more, and more preferably about 98% or more sequence identity tothat particular nucleotide sequence as determined by sequence alignmentprograms described elsewhere herein using default parameters.

[0079] By “variant” protein is intended a protein derived from thenative protein by deletion (so-called truncation) or addition of one ormore amino acids to the N-terminal and/or C-terminal end of the nativeprotein; deletion or addition of one or more amino acids at one or moresites in the native protein; or substitution of one or more amino acidsat one or more sites in the native protein. Variant proteins encompassedby the present invention are biologically active, that is they continueto possess the desired biological activity of the native protein, thatis, modulating DNA metabolism as described herein. Such variants mayresult from, for example, genetic polymorphism or from humanmanipulation. Biologically active variants of a native RPA protein ofthe invention will have at least 40%, 50%, 60%, 70%, generally at least75%, 80%, 85%, preferably about 90% to 95% or more, and more preferablyabout 98% or more sequence identity to the amino acid sequence for thenative protein as determined by sequence alignment programs describedelsewhere herein using default parameters. A biologically active variantof a protein of the invention may differ from that protein by as few as1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, asfew as 4, 3, 2, or even 1 amino acid residue.

[0080] The proteins of the invention may be altered in various waysincluding amino acid substitutions, deletions, truncations, andinsertions. Methods for such manipulations are generally known in theart. For example, amino acid sequence variants of the RPA proteins canbe prepared by mutations in the DNA. Methods for mutagenesis andnucleotide sequence alterations are well known in the art. See, forexample, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel etal. (1987) Methods in Enzymol. 154:367-382; U.S. Pat. No. 4,873,192;Walker and Gaastra, eds. (1983) Techniques in Molecular Biology(MacMillan Publishing Company, New York) and the references citedtherein. Guidance as to appropriate amino acid substitutions that do notaffect biological activity of the protein of interest may be found inthe model of Dayhoff et al. (1978) Atlas of Protein Sequence andStructure (Natl. Biomed. Res. Found., Washington, D.C.), hereinincorporated by reference. Conservative substitutions, such asexchanging one amino acid with another having similar properties, may bepreferred.

[0081] Thus, the genes and nucleotide sequences of the invention includeboth the naturally occurring sequences as well as mutant forms.Likewise, the proteins of the invention encompass both naturallyoccurring proteins as well as variations and modified forms thereof.Such variants will continue to possess the desired activity ininfluencing DNA metabolism. Obviously, the mutations that will be madein the DNA encoding the variant must not place the sequence out ofreading frame and preferably will not create complementary regions thatcould produce secondary mRNA structure. See, EP Patent ApplicationPublication No. 75,444.

[0082] The deletions, insertions, and substitutions of the proteinsequence encompassed herein are not expected to produce radical changesin the characteristics of the protein. However, when it is difficult topredict the exact effect of the substitution, deletion, or insertion inadvance of doing so, one skilled in the art will appreciate that theeffect will be evaluated by routine screening assays. That is, theactivity can be evaluated by assessing DNA binding, recombination,repair and replication. See, for example, Braun et al. (1997)Biochemistry 36:8443-8454; Longhese et al., (1994) Molecular andCellular Biology 14:7884-7890; Stigger et al. (1998) J. Biol. Chem.273:9337-9343; Abremova et al. (1997) Proc. Natl. Acad. Sci. USA94:7186-7191; New et al. (1998) Nature 391:407-410; Bochkareva et al.,(1998) J. Biol. Chem. 273(7):3932-6; Mass et al. (1998) Mol. Cell. Biol.18(11):6399-407; Lavrik et al. (1998) Nucleic Acids Res 26(2):602-7;Sibenaller et al. (1998) 37(36):12496-506; Matsunaga et al. (1996) J.Biol. Chem. 271 (19):11047-50; and Sung (1997) Genes & Development11:1111-21, herein incorporated by reference.

[0083] Variant nucleotide sequences and proteins also encompassnucleotide sequences and proteins derived from a mutagenic andrecombinogenic procedure such as DNA shuffling. With such a procedure,one or more different RPA coding sequences can be manipulated to createa new RPA possessing the desired properties. In this manner, librariesof recombinant polynucleotides are generated from a population ofrelated sequence polynucleotides comprising sequence regions that havesubstantial sequence identity and can be homologously recombined invitro or in vivo. For example, using this approach, sequence motifsencoding a domain of interest may be shuffled between the RPA gene ofthe invention and other known RPA genes to obtain a new gene coding fora protein with an improved property of interest, such as an increasedK_(m) in the case of an enzyme. Strategies for such DNA shuffling areknown in the art. See, for example, Stemmer (1994) Proc. Natl. Acad.Sci. USA 91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri etal. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol.272:336-347; Zhang et al (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509;Crameri et al. (1998) Nature 391:288-291; and U.S. Pat. Nos. 5,605,793and 5,837,458.

[0084] It is recognized that with these nucleotide sequences, antisenseconstructions, complementary to at least a portion of the messenger RNA(mRNA) for the RPA sequences can be constructed. Antisense nucleotidesare constructed to hybridize with the corresponding mRNA. Modificationsof the antisense sequences may be made as long as the sequenceshybridize to and interfere with expression of the corresponding mRNA. Inthis manner, antisense constructions having 70%, preferably 80%, morepreferably 85% sequence similarity to the corresponding antisensesequences may be used. Furthermore, portions of the antisensenucleotides may be used to disrupt the expression of the target gene.Generally, sequences of at least 50 nucleotides, 100 nucleotides, 200nucleotides, or greater may be used.

[0085] The nucleotide sequences of the present invention may also beused in the sense orientation to suppress the expression of endogenousgenes in plants. Methods for suppressing gene expression in plants usingnucleotide sequences in the sense orientation are known in the art. Themethods generally involve transforming plants with a DNA constructcomprising a promoter that drives expression in a plant operably linkedto at least a portion of a nucleotide sequence that corresponds to thetranscript of the endogenous gene. Typically, such a nucleotide sequencehas substantial sequence identity to the sequence of the transcript ofthe endogenous gene, preferably greater than about 65% sequenceidentity, more preferably greater than about 85% sequence identity, mostpreferably greater than about 95% sequence identity. See, U.S. Pat. Nos.5,283,184 and 5,034,323; herein incorporated by reference.

[0086] Use of the polypeptides and proteins, and fragments and variantsthereof, for producing antibodies are also encompassed by the invention.The invention also encompasses using such antibodies to determine RPAprotein levels, and to modulate one or more biological activities orinteractions of RPA. Methods for the production of antibodies are knownin the art. See, for example, Harlow and Lane, antibodies, A LaboratoryManual, Cold Spring Harbor Publications, New York (1988); and thereference is cited therein.

[0087] The RPA sequences of the invention may be optimized for enhancedexpression in plants of interest. See, for example, EPA0359472;WO91/16432; Perlak et al. (1991) Proc. Natl. Acad. Sci. USA88:3324-3328; and Murray et al. (1989) Nucleic Acids Res. 17:477-498. Inthis manner, the genes can be synthesized utilizing plant-preferredcodons. See, for example, Murray et al. (1989) Nucleic Acids Res.17:477-498, the disclosure of which is incorporated herein by reference.In this manner, synthetic genes can also be made based on thedistribution of codons a particular host uses for a particular aminoacid. Thus, the nucleotide sequences can be optimized for expression inany plant. It is recognized that all or any part of the gene sequencemay be optimized or synthetic. That is, synthetic or partially optimizedsequences may also be used.

[0088] Thus nucleotide sequences of the invention and the proteinsencoded thereby include the native forms as well as variants thereof.The variant proteins will be substantially homologous and functionallyequivalent to the native proteins. A variant of a native protein is“substantially homologous” to the native protein when at least about80%, more preferably at least about 90%, and most preferably at leastabout 95% of its amino acid sequence is identical to the amino acidsequence of the native protein. By “functionally equivalent” is intendedthat the sequence of the variant defines a chain that produces a proteinhaving substantially the same biological effect as the native protein ofinterest. Such functionally equivalent variants that comprisesubstantial sequence variations are also encompassed by the invention.

[0089] The nucleotide sequences of the invention can be used to isolatecorresponding sequences from other organisms, particularly other plants,more particularly other monocots. In this manner, methods such as PCR,hybridization, and the like can be used to identify such sequences basedon their sequence homology to the sequence set forth herein. Sequencesisolated based on their sequence identity to the entire RPA sequencesset forth herein or to fragments thereof are encompassed by the presentinvention.

[0090] In a PCR approach, oligonucleotide primers can be designed foruse in PCR reactions to amplify corresponding DNA sequences from cDNA orgenomic DNA extracted from any plant of interest. Methods for designingPCR primers and PCR cloning are generally known in the art and aredisclosed in Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).See also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods andApplications (Academic Press, New York); Innis and Gelfand, eds. (1995)PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds.(1999) PCR Methods Manual (Academic Press, New York). Known methods ofPCR include, but are not limited to, methods using paired primers,nested primers, single specific primers, degenerate primers,gene-specific primers, vector-specific primers, partially-mismatchedprimers, and the like.

[0091] In hybridization techniques, all or part of a known nucleotidesequence is used as a probe that selectively hybridizes to othercorresponding nucleotide sequences present in a population of clonedgenomic DNA fragments or cDNA fragments (i.e., genomic or cDNAlibraries) from a chosen organism. The hybridization probes may begenomic DNA fragments, cDNA fragments, RNA fragments, or otheroligonucleotides, and may be labeled with a detectable group such as³²P, or any other detectable marker. Thus, for example, probes forhybridization can be made by labeling synthetic oligonucleotides basedon the RPA sequences of the invention. Methods for preparation of probesfor hybridization and for construction of cDNA and genomic-libraries aregenerally known in the art and are disclosed in Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Plainview, N.Y.).

[0092] For example, the entire RPA sequence disclosed herein, or one ormore portions thereof, may be used as a probe capable of specificallyhybridizing to corresponding RPA sequences and messenger RNAs. Toachieve specific hybridization under a variety of conditions, suchprobes include sequences that are unique among RPA sequences and arepreferably at least about 10 nucleotides in length, and most preferablyat least about 20 nucleotides in length. Such probes may be used toamplify corresponding RPA sequences from a chosen plant by PCR. Thistechnique may be used to isolate additional coding sequences from adesired plant or as a diagnostic assay to determine the presence ofcoding sequences in a plant Hybridization techniques includehybridization screening of plated DNA libraries (either plaques orcolonies; see, for example, Sambrook et al. (1989) Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y.).

[0093] Hybridization of such sequences may be carried out understringent conditions. By “stringent conditions” or “stringenthybridization conditions” is intended conditions under which a probewill hybridize to its target sequence to a detectably greater degreethan to other sequences (e.g., at least 2-fold over background).Stringent conditions are sequence-dependent and will be different indifferent circumstances. By controlling the stringency of thehybridization and/or washing conditions, target sequences that are 100%complementary to the probe can be identified (homologous probing).Alternatively, stringency conditions can be adjusted to allow somemismatching in sequences so that lower degrees of similarity aredetected (heterologous probing). Generally, a probe is less than about1000 nucleotides in length, preferably less than 500 nucleotides inlength.

[0094] Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1×to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., anda wash in 0.5×to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C.

[0095] Specificity is typically the function of post-hybridizationwashes, the critical factors being the ionic strength and temperature ofthe final wash solution. For DNA-DNA hybrids, the T_(m) can beapproximated from the equation of Meinkoth and Wahl (1984) Anal.Biochem. 138:267-284: T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)-0.61 (%form)-500/L; where M is the molarity of monovalent cations, % GC is thepercentage of guanosine and cytosine nucleotides in the DNA, % form isthe percentage of formamide in the hybridization solution, and L is thelength of the hybrid in base pairs. The T_(m) is the temperature (underdefined ionic strength and pH) at which 50% of a complementary targetsequence hybridizes to a perfectly matched probe. T_(m) is reduced byabout 1° C. for each 1% of mismatching; thus, T_(m), hybridization,and/or wash conditions can be adjusted to hybridize to sequences of thedesired identity. For example, if sequences with ≧90% identity aresought, the T_(m) can be decreased 10° C. Generally, stringentconditions are selected to be about 5° C. lower than the thermal meltingpoint (T_(m)) for the specific sequence and its complement at a definedionic strength and pH. However, severely stringent conditions canutilize a hybridization and/or wash at 1, 2, 3, or 4° C. lower than thethermal melting point (T_(m)); moderately stringent conditions canutilize a hybridization and/or wash at 6, 7, 8, 9, or 10° C. lower thanthe thermal melting point (T_(m)); low stringency conditions can utilizea hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower thanthe thermal melting point (T_(m)). Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m) of less than 45°C (aqueous solution) or32° C. (formamide solution), it is preferred to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen (1993)Laboratory Techniques in Biochemistry and MolecularBiology-Hybridization with Nucleic Acid Probes, Part I, Chapter 2(Elsevier, N.Y.); and Ausubel et al., eds. (1995) Current Protocols inMolecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience,New York). See Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).

[0096] Thus, isolated sequences that have promoter activity or encodefor a RPA protein and which hybridize under stringent conditions to theRPA sequences disclosed herein, or to fragments thereof, are encompassedby the present invention. Such sequences will be at least 40% to 50%homologous, about 60% to 70% homologous, and even about 75%, 80%, 85%,90%, 95% to 98% homologous or more with the disclosed sequences. Thatis, the sequence identity of sequences may range, sharing at least 40%to 50%, about 60% to 70%, and even about 75%, 80%, 85%, 90%, 95% to 98%or more sequence identity.

[0097] The following terms are used to describe the sequencerelationships between two or more nucleic acids or polynucleotides: (a)“reference sequence”, (b) “comparison window”, (c) “sequence identity”,(d) “percentage of sequence identity”, and (e) “substantial identity”.

[0098] (a) As used herein, “reference sequence” is a defined sequenceused as a basis for sequence comparison. A reference sequence may be asubset or the entirety of a specified sequence; for example, as asegment of a full-length cDNA or gene sequence, or the complete cDNA orgene sequence.

[0099] (b) As used herein, “comparison window” makes reference to acontiguous and specified segment of a polynucleotide sequence, whereinthe polynucleotide sequence in the comparison window may compriseadditions or deletions (i.e., gaps) compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. Generally, the comparison window is at least 20contiguous nucleotides in length, and optionally can be 30, 40, 50, 100,or longer. Those of skill in the art understand that to avoid a highsimilarity to a reference sequence due to inclusion of gaps in thepolynucleotide sequence a gap penalty is typically introduced and issubtracted from the number of matches.

[0100] Methods of alignment of sequences for comparison are well knownin the art. Thus, the determination of percent identity between any twosequences can be accomplished using a mathematical algorithm. Preferred,non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller (1988) CABIOS 4:11-17; the local homology algorithmof Smith et al., (1981) Adv. Appl. Math. 2:482; the homology alignmentalgorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; thesearch-for-similarity-method of Pearson and Lipman (1988) Proc. Natl.Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul (1990)Proc. Natl. Acad. Sci. USA 872264, modified as in Karlin and Altschul(1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

[0101] Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the Wisconsin Genetics Software Package, Version 8 (availablefrom Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis.,USA). Alignments using these programs can be performed using the defaultparameters. The CLUSTAL program is well described by Higgins et al.(1988) Gene 73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153;Corpet et a. (1988) Nucleic Acids Res. 16:10881-90; Huang et a. (1992)CABIOS 8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331.The ALIGN program is based on the algorithm of Myers and Miller (1988)supra. A PAM120 weight residue table, a gap length penalty of 12, and agap penalty of 4 can be used with the ALIGN program when comparing aminoacid sequences. The BLAST programs of Altschul et al (1990) J. Mol.Biol. 215:403 are based on the algorithm of Karlin and Altschul (1990)supra. BLAST nucleotide searches can be performed with the BLASTNprogram, score=100, wordlength=12, to obtain nucleotide sequenceshomologous to a nucleotide sequence encoding a protein of the invention.BLAST protein searches can be performed with the BLASTX program,score=50, wordlength=3, to obtain amino acid sequences homologous to aprotein or polypeptide of the invention. To obtain gapped alignments forcomparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25:3389.Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform aniterated search that detects distant relationships between molecules.See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST,PSI-BLAST, the default parameters of the respective programs (e.g.,BLASTN for nucleotide sequences, BLASTX for proteins) can be used. Seewww.ncbi.nim.nih.ov. Alignment may also be performed manually byinspection. Alignment may also be performed manually by inspection.

[0102] For purposes of the present invention, comparison of nucleotideor protein sequences for determination of percent sequence identity tothe RPA sequences disclosed herein is preferably made using the GCGPileUp program, version 10.00, with its default parameters or anyequivalent program. By “equivalent program” is intended any sequencecomparison program that, for any two sequences in question, generates analignment having identical nucleotide or amino acid residue matches andan identical percent sequence identity when compared to thecorresponding alignment generated by the preferred program.

[0103] (c) As used herein, “sequence identity” or “identity” in thecontext of two nucleic acid or polypeptide sequences makes reference tothe residues in the two sequences that are the same when aligned formaximum correspondence over a specified comparison window. Whenpercentage of sequence identity is used in reference to proteins it isrecognized that residue positions which are not identical often differby conservative amino acid substitutions, where amino acid residues aresubstituted for other amino acid residues with similar chemicalproperties (e.g., charge or hydrophobicity) and therefore do not changethe functional properties of the molecule. When sequences differ inconservative substitutions, the percent sequence identity may beadjusted upwards to correct for the conservative nature of thesubstitution. Sequences that differ by such conservative substitutionsare said to have “sequence similarity” or “similarity”. Means for makingthis adjustment are well known to those of skill in the art. Typicallythis involves scoring a conservative substitution as a partial ratherthan a full mismatch, thereby increasing the percentage sequenceidentity. Thus, for example, where an identical amino acid is given ascore of 1 and a non-conservative substitution is given a score of zero,a conservative substitution is given a score between zero and 1. Thescoring of conservative substitutions is calculated, e.g., asimplemented in the program PC/GENE (Intelligenetics, Mountain View,Calif.).

[0104] (d) As used herein, “percentage of sequence identity” means thevalue determined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

[0105] (e)(i) The term “substantial identity” of polynucleotidesequences means that a polynucleotide comprises a sequence that has atleast 70% sequence identity, preferably at least 80%, more preferably atleast 90%, and most preferably at least 95%, compared to a referencesequence using one of the alignment programs described using standardparameters. One of skill in the art will recognize that these values canbe appropriately adjusted to determine corresponding identity ofproteins encoded by two nucleotide sequences by taking into accountcodon degeneracy, amino acid similarity, reading frame positioning, andthe like. Substantial identity of amino acid sequences for thesepurposes normally means sequence identity of at least 60%, morepreferably at least 70%, 80%, 90%, and most preferably at least 95%.

[0106] Another indication that nucleotide sequences are substantiallyidentical is if two molecules hybridize to each other under stringentconditions. Generally, stringent conditions are selected to be about 5°C. lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH. However, stringentconditions encompass temperatures in the range of about 1° C. to about20° C., depending upon the desired degree of stringency as otherwisequalified herein. Nucleic acids that do not hybridize to each otherunder stringent conditions are still substantially identical if thepolypeptides they encode are substantially identical. This may occur,e.g., when a copy of a nucleic acid is created using the maximum codondegeneracy permitted by the genetic code. One indication that twonucleic acid sequences are substantially identical is when thepolypeptide encoded by the first nucleic acid is immunologically crossreactive with the polypeptide encoded by the second nucleic acid.

[0107] (e)(ii) The term “substantial identity” in the context of apeptide indicates that a peptide comprises a sequence with at least 70%sequence identity to a reference sequence, preferably 80%, morepreferably 85%, most preferably at least 90% or 95% sequence identity tothe reference sequence over a specified comparison window. Preferably,optimal alignment is conducted using the homology alignment algorithm ofNeedleman et al. (1970) J. Mol. Biol. 48:443. An indication that twopeptide sequences are substantially identical is that one peptide isimmunologically reactive with antibodies raised against the secondpeptide. Thus, a peptide is substantially identical to a second peptide,for example, where the two peptides differ only by a conservativesubstitution. Peptides that are “substantially similar” share sequencesas noted above except that residue positions that are not identical maydiffer by conservative amino acid changes.

[0108] Using the nucleic acids of the present invention, one may expressa protein of the present invention in a recombinantly engineered cellsuch as bacteria, yeast, insect, mammalian, or preferably plant cells.The cells produce the protein in a non-natural condition (e.g., inquantity, composition, location, and/or time), because they have beengenetically altered through human intervention to do so.

[0109] It is expected that those of skill in the art are knowledgeablein the numerous expression systems available for expression of a nucleicacid encoding a protein of the present invention. No attempt to describein detail the various methods known for the expression of proteins inprokaryotes or eukaryotes will be made.

[0110] In brief summary, the expression of isolated nucleic acidsencoding a protein of the present invention will typically be achievedby operably linking, for example, the DNA or cDNA to a promoter (whichis either constitutive or inducible), followed by incorporation into anexpression vector. The vectors can be suitable for replication andintegration in either prokaryotes or eukaryotes. Typical expressionvectors contain transcription and translation terminators, initiationsequences, and promoters useful for regulation of the expression of theDNA encoding a protein of the present invention. To obtain high levelexpression of a cloned gene, it is desirable to construct expressionvectors which contain, at the minimum, a strong promoter to directtranscription, a ribosome binding site for translational initiation, anda transcription/translation terminator. One of skill would recognizethat modifications can be made to a protein of the present inventionwithout diminishing its biological activity. Some modifications may bemade to facilitate the cloning, expression, or incorporation of thetargeting molecule into a fusion protein. Such modifications are wellknown to those of skill in the art and include, for example, amethionine added at the amino terminus to provide an initiation site, oradditional amino acids (e.g., poly His) placed on either terminus tocreate conveniently located restriction sites or termination codons orpurification sequences.

[0111] Prokaryotic cells may be used as hosts for expression.Prokaryotes most frequently are represented by various strains of E.coli; however, other microbial strains may also be used. Commonly usedprokaryotic control sequences which are defined herein to includepromoters for transcription initiation, optionally with an operator,along with ribosome binding site sequences, include such commonly usedpromoters as the beta lactamase (penicillinase) and lactose (lac)promoter systems (Chang et al. (1977) Nature 198:1056), the tryptophan(trp) promoter system (Goeddel et al. (1980) Nucleic Acids Res. 8:4057)and the lambda-derived P L promoter and N-gene ribosome binding site(Shimatake et al. (1981) Nature 292:128). The inclusion of selectionmarkers in DNA vectors transfected in E. coli is also useful. Examplesof such markers include genes specifying resistance to ampicillin,tetracycline, or chloramphenicol.

[0112] The vector is selected to allow introduction into the appropriatehost cell. Bacterial vectors are typically of plasmid or phage origin.Appropriate bacterial cells are infected with phage vector particles ortransfected with naked phage vector DNA. If a plasmid vector is used,the bacterial cells are transfected with the plasmid vector DNA.Expression systems for expressing a protein of the present invention areavailable using Bacillus sp. and Salmonella (Palva et al. (1983) Gene22:229-235; Mosbach et al. (1983) Nature 302:543-545).

[0113] A variety of eukaryotic expression systems such as yeast, insectcell lines, plant and mammalian cells, are known to those of skill inthe art. The sequences of the present invention can be expressed inthese eukaryotic systems. In some embodiments, transformed/transfectedplant cells are employed as expression systems for production of theproteins of the instant invention.

[0114] Synthesis of heterologous proteins in yeast is well known.Sherman, F. et al. (1982) Methods in Yeast Genetics, Cold Spring HarborLaboratory is a well recognized work describing the various methodsavailable to produce the protein in yeast. Two widely utilized yeast forproduction of eukaryotic proteins are Saccharomyces cerevisia and Pichiapastoris. Vectors, strains, and protocols for expression inSaccharomyces and Pichia are known in the art and available fromcommercial suppliers (e.g., Invitrogen). Suitable vectors usually haveexpression control sequences, such as promoters, including3-phosphoglycerate kinase or alcohol oxidase, and an origin ofreplication, termination sequences and the like as desired.

[0115] A protein of the present invention, once expressed, can beisolated from yeast by lysing the cells and applying standard proteinisolation techniques to the lysates. The monitoring of the purificationprocess can be accomplished by using Western blot techniques orradioimmunoassay of other standard immunoassay techniques.

[0116] The sequences encoding proteins of the present invention can alsobe ligated to various expression vectors for use in transfecting cellcultures of, for instance, mammalian, insect, or plant origin.Illustrative of cell cultures useful for the production of the peptidesare mammalian cells. Mammalian cell systems often will be in the form ofmonolayers of cells although mammalian cell suspensions may also beused. A number of suitable host cell lines capable of expressing intactproteins have been developed in the art, and include the HEK293, BHK21,and CHO cell lines. Expression vectors for these cells can includeexpression control sequences, such as an origin of replication, apromoter (e.g., the CMV promoter, a HSV tk promoter or pgk(phosphoglycerate kinase promoter)), an enhancer (Queen et al. (1986)Immunol. Rev. 89:49), and necessary processing information sites, suchas ribosome binding sites, RNA splice sites, polyadenylation sites(e.g., an SV40 large T Ag poly A addition site), and transcriptionalterminator sequences. Other animal cells useful for production ofproteins of the present invention are available, for instance, from theAmerican Type Culture Collection Catalogue of Cell Lines and Hybridomas(7th edition, 1992).

[0117] Appropriate vectors for expressing proteins of the presentinvention in insect cells are usually derived from the SF9 baculovirus.Suitable insect cell lines include mosquito larvae, silkworm, armyworm,moth and Drosophila cell lines such as a Schneider cell line (SeeSchneider et al. (1987) J. Embryol. Exp. Morphol. 27: 353-365).

[0118] As with yeast, when higher animal or plant host cells areemployed, polyadenylation or transcription terminator sequences aretypically incorporated into the vector. An example of a terminatorsequence is the polyadenylation sequence from the bovine growth hormonegene. Sequences for accurate splicing of the transcript may also beincluded. An example of a splicing sequence is the VP1 intron from SV40(Sprague et al. (1983) J. Virol. 45:773-781). Additionally, genesequences to control replication in the host cell may be incorporatedinto the vector such as those found in bovine papilloma virus-typevectors. Saveria-Campo, M., Bovine Papilloma Virus DNA a EukaryoticCloning Vector in DNA Cloning Vol. II a Practical Approach, D. M.Glover, ed., IRL Press, Arlington, Va. pp. 213-238 (1985).

[0119] The sequences of the invention can be introduced into any plantof interest, and used for transformation of any plant species. Thesequences to be introduced may be used in expression cassettes forexpression in the particular plant of interest.

[0120] Plants of interest include, but are not limited to corn (Zeamays), Brassica sp. (e.g., B. napus, B. rapa, B. juncea), particularlythose Brassica species useful as sources of seed oil, alfalfa (Medicagosativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghumbicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetumglaucum), proso millet (Panicum miliaceum), foxtail millet (Setariaitalica), finger millet (Eleusine coracana)), sunflower (Helianthusannuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum),soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanumtuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense,Gossypium hirsutum), sweet potato (Ipomoea batatus), cassaya (Manihotesculenta), coffee (Cofea spp.), coconut (Cocos nucifera), pineapple(Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao),tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana),fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica),olive (Olea europaea), papaya (Carica papaya), cashew (Anacardiumoccidentale), macadamia (Macadamia integrifolia), almond (Prunusamygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.),oats, barley, vegetables, ornamentals, and conifers.

[0121] Vegetables include tomatoes (Lycopersicon esculentum), lettuce(e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima beans(Phaseolus limensis), peas (Lathyrus spp.), and members of the genusCucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis),and musk melon (C. melo). Ornamentals include azalea (Rhododendronspp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscusrosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils(Narcissus spp.), petunias (Petunia hybrida), carnation (Dianthuscaryophyllus), poinsettia (Euphorbia pulcherrima), and chrysanthemum.Conifers that may be employed in practicing the present inventioninclude, for example, pines such as loblolly pine (Pinus taeda), slashpine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine(Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir(Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitkaspruce (Picea glauca); redwood (Sequoia sempervirens); true firs such assilver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedarssuch as Western red cedar (Thuja plicata) and Alaska yellow-cedar(Chamaecyparis nootkatensis). Preferably, plants of the presentinvention are crop plants (for example, corn, alfalfa, sunflower,Brassica, soybean, cotton, safflower, peanut, sorghum, wheat, millet,tobacco, etc.), more preferably corn and soybean plants, yet morepreferably corn plants.

[0122] Plants of particular interest include grain plants that provideseeds of interest, oil-seed plants, and leguminous plants. Seeds ofinterest include grain seeds, such as corn, wheat, barley, rice,sorghum, rye, etc. Oil-seed plants include cotton, soybean, safflower,sunflower, Brassica, maize, alfalfa, palm, coconut, etc. Leguminousplants include beans and peas. Beans include guar, locust bean,fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, favabean, lentils, chickpea, etc.

[0123] The RPA coding and antisense sequences of the invention areprovided in expression cassettes for expression in the plant ofinterest. The cassette will include 5′ and 3′ regulatory sequencesoperably linked to a RPA sequence of the invention. The cassette mayadditionally contain at least one additional gene to be cotransformedinto the organism. Alternatively, the additional gene(s) can be providedon another expression cassette. By “operably linked” is intended afunctional linkage between a promoter and a second sequence, wherein thepromoter sequence initiates and mediates transcription of the DNAsequence corresponding to the second sequence. Generally, operablylinked means that the nucleic acid sequences being linked are contiguousand, where necessary to join two protein coding regions, contiguous andin the same reading frame.

[0124] Such an expression cassette is provided with a plurality ofrestriction sites for insertion of the RPA sequence to be under thetranscriptional regulation of the regulatory regions. The expressioncassette may additionally contain selectable marker genes.

[0125] The expression cassette will include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region, aRPA DNA sequence of the invention, and a transcriptional andtranslational termination region functional in plants. Thetranscriptional initiation region, the promoter, may be native oranalogous or foreign or heterologous to the plant host. Additionally,the promoter may be the natural sequence or alternatively a syntheticsequence. By “foreign” is intended that the transcriptional initiationregion is not found in the native plant into which the transcriptionalinitiation region is introduced. As used herein, a chimeric genecomprises a coding sequence operably linked to a transcriptioninitiation region that is heterologous to the coding sequence.

[0126] While it may be preferable to express the sequences usingheterologous promoters, the native promoter sequences may be used. Suchconstructs would change expression levels of RPA in the plant or plantcell. Thus, the phenotype of the plant or plant cell is altered.

[0127] The termination region may be native with the transcriptionalinitiation region, may be native with the operably linked DNA sequenceof interest, or may be derived from another source. Convenienttermination regions are available from the Ti-plasmid of A. tumefaciens,such as the octopine synthase and nopaline synthase termination regions.See also Guerineau et al., (1991) Mol. Gen. Genet 262:141-144; Proudfoot(1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149;Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; andJoshi et al. (1987) Nucleic Acid Res. 15:9627-9639.

[0128] Where appropriate, the gene(s) may be optimized for increasedexpression in the transformed plant. That is, the genes can besynthesized using plant-preferred codons for improved expression. See,for example, Campbell and Gowri (1990) Plant Physiol. 92:1-11 for adiscussion of host-preferred codon usage. Methods are available in theart for synthesizing plant-preferred genes. See, for example, U.S. Pat.No. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic AcidsRes. 17:477-498, herein incorporated by reference.

[0129] Additional sequence modifications are known to enhance geneexpression in a cellular host. These include elimination of sequencesencoding spurious polyadenylation signals, exon-intron splice sitesignals, transposon-like repeats, and other such well-characterizedsequences that may be deleterious to gene expression. The G-C content ofthe sequence may be adjusted to levels average for a given cellularhost, as calculated by reference to known genes expressed in the hostcell. When possible, the sequence is modified to avoid predicted hairpinsecondary mRNA structures.

[0130] The expression cassettes may additionally contain 5′ leadersequences in the expression cassette construct. Such leader sequencescan act to enhance translation. Translation leaders are known in the artand include: picornavirus leaders, for example, EMCV leader(Encephalomyocarditis 5′ noncoding region) (Elroy-Stein et al., (1989)PNAS USA 86:6126-6130); potyvirus leaders, for example, TEV leader(Tobacco Etch Virus) (Allison et al. (1986); MDMV leader (Maize DwarfMosaic Virus); Virology 154:9-20), and human immunoglobulin heavy-chainbinding protein (BiP), (Macejak et al. (1991) Nature 353:90-94);untranslated leader from the coat protein mRNA of alfalfa mosaic virus(AMV RNA 4) (Jobling et al. (1987) Nature 325:622-625); tobacco mosaicvirus leader (TMV) (Gallie et al. (1989) in Molecular Biology of RNA,ed. Cech (Liss, New York), pp. 237-256); and maize chlorotic mottlevirus leader (MCMV) (Lommel et al. (1991) Virology 81:382-385). Seealso, Della-Cioppa et al. (1987) Plant Physiol. 84:965-968. Othermethods known to enhance translation can also be utilized, for example,introns, and the like.

[0131] In preparing the expression cassette, the various DNA fragmentsmay be manipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

[0132] The sequences of the present invention can be used to transformor transfect any plant. In this manner, genetically modified plants,plant cells, plant tissue, seed, and the like can be obtained.Transformation protocols as well as protocols for introducing nucleotidesequences into plants may vary depending on the type of plant or plantcell, i.e., monocot or dicot, targeted for transformation. Suitablemethods of introducing nucleotide sequences into plant cells andsubsequent insertion into the plant genome include microinjection(Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggset al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606,Agrobacterium-mediated transformation (Townsend et al., U.S. Pat. No.5,563,055), direct gene transfer (Paszkowski et al. (1984) EMBO J.3:2717-2722), and ballistic particle acceleration (see, for example,Sanford et al., U.S. Pat. No. 4,945,050; Tomes et al., U.S. Pat. No.5,879,918; Tomes et al., U.S. Pat. No. 5,886,244; Bidney et al., U.S.Pat. No. 5,932,782; Tomes et al. (1995) “Direct DNA Transfer into IntactPlant Cells via Microprojectile Bombardment,” in Plant Cell, Tissue, andOrgan Culture: Fundamental Methods, ed. Gamborg and Phillips(Springer-Verlag, Berlin); and McCabe et al. (1988) Biotechnology6:923-926). Also see Weissinger et al. (1988) Ann. Rev. Genet22:421-477; Sanford et al., (1987) Particulate Science and Technology5:27-37 (onion); Christou et al. (1988) Plant Physiol. 87:671-674(soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol.27P:175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet 96:319-324(soybean); Datta et al. (1990) Biotechnology 8:736-740 (rice); Klein etal. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein et al.(1988) Biotechnology 6:559-563 (maize); Tomes, U.S. Pat. No. 5,240,855;Buising et al., U.S. Pat. Nos. 5,322,783 and 5,324,646; Klein et al.(1988) Plant Physiol. 91:440-444 (maize); Fromm et al. (1990)Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al. (1984)Nature (London) 311:763-764; Bowen et al., U.S. Pat. No. 5,736,369(cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA84:5345-5349 (Liliaceae); De Wet et al. (1985) in The ExperimentalManipulation of Ovule Tissues, ed. Chapman et al. (Longman, N.Y.), pp.197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566(whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell4:1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413(rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize viaAgrobacterium tumefaciens); all of which are herein incorporated byreference.

[0133] The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting hybrid having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that expression of the desired phenotypic characteristicis stably maintained and inherited and then seeds harvested to ensureexpression of the desired phenotypic characteristic has been achieved.

[0134] Transgenic plants expressing the selectable marker can bescreened for transmission of the nucleic acid of the present inventionby, for example, standard immunoblot and DNA detection techniques.Transgenic lines are also typically evaluated on levels of expression ofthe heterologous nucleic acid. Expression at the RNA level can bedetermined initially to identify and quantitate expression-positiveplants. Standard techniques for RNA analysis can be employed and includePCR amplification assays using oligonucleotide primers designed toamplify only the heterologous RNA templates and solution hybridizationassays using heterologous nucleic acid-specific probes. The RNA-positiveplants can then be analyzed for protein expression by Western immunoblotanalysis using the specifically reactive antibodies of the presentinvention. In addition, in situ hybridization and immunocytochemistryaccording to standard protocols can be done using heterologous nucleicacid specific polynucleotide probes and antibodies, respectively, tolocalize sites of expression within transgenic tissue. Generally, anumber of transgenic lines are usually screened for the incorporatednucleic acid to identify and select plants with the most appropriateexpression profiles.

[0135] A preferred embodiment is a transgenic plant that is homozygousfor the added heterologous nucleic acid; i.e., a transgenic plant thatcontains two added nucleic acid sequences, one gene at the same locus oneach chromosome of a chromosome pair. A homozygous transgenic plant canbe obtained by sexually mating (selfing) a heterozygous transgenic plantthat contains a single added heterologous nucleic acid, germinating someof the seed produced and analyzing the resulting plants produced foraltered RPA expression relative to a control plant (i.e., native,non-transgenic). Backcrossing to a parental plant and out-crossing witha non-transgenic plant are also contemplated.

[0136] The present invention further provides a method for modulating(i.e., increasing or decreasing) RPA levels in a plant or part thereof.Modulation can be effected by increasing or decreasing the total amountof RPA (i.e., its content) and/or the ratio of various RPA subunitproteins (i.e., its composition) in the plant. The method comprisestransforming a plant cell with a recombinant expression cassettecomprising a polynucleotide of the present invention as described aboveto obtain a transformed plant cell, growing the transformed plant cellunder plant forming conditions, and inducing expression of apolynucleotide of the present invention in the plant for a timesufficient to modulate RPA content and/or composition in the plant orplant part.

[0137] In some embodiments, RPA in a plant may be modulated by altering,in vivo or in vitro, the promoter of a non-isolated RPA gene to up- ordown-regulate gene expression. In some embodiments, the coding regionsof native RPA genes an be altered via substitution, addition, insertion,or deletion to decrease activity of the encoded enzyme. See, e.g.,Kmiec, U.S. Pat. No. 5,565,350; Zarling et al., PCT/US93/03868. And insome embodiments, an isolated nucleic acid (e.g., a vector) comprising apromoter sequence is transfected into a plant cell. Subsequently, aplant cell comprising the promoter operably linked to a polynucleotideof the present invention is selected by means known to those of skill inthe art such as, but not limited to, Southern blot, DNA sequencing, orPCR analysis using primers specific to the promoter and to the gene anddetecting amplicons produced therefrom. A plant or plant part altered ormodified by the foregoing embodiments is grown under plant formingconditions for a time sufficient to modulate RPA content and/orcomposition in the plant. Plant forming conditions are well known in theart and discussed briefly, supra.

[0138] In general, content or composition is increased or decreased byat least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% relative toa native control plant, plant part, or cell lacking the aforementionedrecombinant expression cassette. Modulation in the present invention mayoccur during and/or subsequent to growth of the plant to the desiredstage of development. Modulating nucleic acid expression temporallyand/or in particular tissues can be controlled by employing theappropriate promoter operably linked to a polynucleotide of the presentinvention in, for example, sense or antisense orientation as discussedin greater detail, supra. Induction of expression of a polynucleotide ofthe present invention can also be controlled by exogenous administrationof an effective amount of inducing compound. Inducible promoters andinducing compounds that activate expression from these promoters arewell known in the art. In preferred embodiments, RPA is modulated inmonocots, particularly maize.

[0139] The ability of RPA to interact with multiple proteins or proteincomplexes allows it to participate and regulate these multiple pathwaysof DNA metabolism. For example, it has been shown in mammalian systemsthat are RPA interacts with DNA polymerase alpha (Barun et al. (1997)Biochemistry 36:8443-8454), p53 (Dutta et al. (1993) Nature 365:79-82),RAD 62 (Park et al. (1996) J. Biol. Chem. 271:18996-19000).

[0140] Participation of the middle subunit of RPA in protein-proteininteractions has also been shown. Examples of such interactions include,but are not limited to interactions with XPA protein and RAD 52 (He etal. (1995) Nature 374:566-69; Matsuda et al. (1995) J. Biol. Chem.270:4152-57; Li et al. (1995) Mol. Cell. Biol. 15:5396-402, Park et al.(1996) J. Biol. Chem. 271:18996-19000); and PCNA (Shivji et al. (1995)Biochemistry 34:5011-5017).

[0141] Similarly, yeast RPA has been shown to be involved in multiplefunctions in DNA metabolism (Umezu et al. (1998) Genetics 148:989-1005).Therefore, the proteins of the invention may be useful as a ligand topurify and clone other proteins involved in DNA recombination, repair,and replication. Particularly, the maize proteins may be useful topurify other maize proteins involved in DNA metabolism. For example, theRPA proteins of the invention may be insolubilized on a solid matrix(e.g. agrose or nylon beads) for affinity purification, or the RPA cDNAmay be used as a bait in a yeast to-hybrid system. In this manner, otherproteins may be used identified and isolated.

[0142] The following examples are offered by way of illustration and notby way of limitation.

EXPERIMENTAL Example 1 cDNA Cloning

[0143] Total RNA was isolated from corn tissues with TRIzol Reagent(Life Technology, Inc. Gaithersburg, Md.) using a modification of theguanidine isothiocyanate/acid-phenol procedure described by Chomczynskiand Sacchi (Chomczynski et al. (1987) Anal. Biochem. 162:156). In brief,plant tissue samples were pulverized in liquid nitrogen before theaddition of the TRIzol Reagent, and then were further homogenized with amortar and pestle. Addition of chloroform by centrifugation wasconducted for separation of an aqueous phase and an organic phase. Thetotal RNA was recovered by precipitation with isopropyl alcohol from theaqueous phase.

[0144] The selection of poly(A)+ RNA from total RNA was performed usingPolyATract system (Promega Corporation, Madison, Wis.). In brief,biotinylated oligo (dT) primers were used to hybridize to the 3′ poly(A)tails on mRNA. The hybrids were captured using streptavidin coupled toparamagnetic particles and a magnetic separation stand. The mRNA waswashed at high stringent condition and cluted by Rnase-free deionizedwater.

[0145] Synthesis of the cDNA was performed and unidirectional cDNAlibraries were constructed using the SuperScript Plasmid System (LifeTechnology, Inc., Gaithersburg, Md.). First strand of cDNA wassynthesized by priming an oligo(dT) primer containing a Not I site. Thereaction was catalyzed by SuperScript Reverse Transcriptase II at 45° C.The second strand of cDNA was labeled with α-³²P-dCTP and portions ofthe molecules smaller than 500 base pairs and unligated adapters wereremoved by Sephacryl-S400 chromatography. The selected cDNA moleculeswere ligated into pSPORT1 reference vector between the Not I and Sal Isites.

[0146] Individual colonies were picked and DNA was prepared either byPCR with M13 forward primers and M13 reverse primers, or by plasmidminiprep isolation. All the cDNA clones were sequenced using M13 reverseprimers.

[0147] Two maize homologues for RPA large subunit (ZmRPALSH) have beenisolated. The genes map to two different chromosomes as shown below inTable 1. The amino acid and nucleotide sequences for the two homologuesare set forth in SEQ ID NOs: 1-4. TABLE 1 Maize RPA Large Subunit GenesMap to Two Different Chromosomes Clone ID Chromosome No. HomologueCBPBS68 c9 ZmRPALSH1 CCRBJ83 c9 ZmRPALSH1 CDPGS47 c9 ZmRPALSH1 CHCLE65c9 ZmRPALSH1 CJLPL35 c9 ZmRPALSH1 COMGE67 c9 ZmRPALSH1 CBAAK06 c9ZmRPALSH2 CDPGS46 c9 ZmRPALSH2 CERAG93 c9 ZmRPALSH2 COMFY67 c9 ZmRPALSH2

[0148] Ten ESTs, which form two different contigs for maize RPA largesubunit, were used as probes for mapping experiments. Each contigrepresents one maize homologue for RPALS.

[0149] Seven maize homologues for RPA middle subunit (ZmRPAMSH) havebeen isolated. The genes map to chromosomes 5 as shown below in Table 2.The nucleotide and amino acid sequences of the seven homologues are setforth in SEQ ID NOs: 11-22. TABLE 2 Maize Homologues of EukaryoticReplication Protein A Middle Subunit Map lone ID Homologue LibraryPosition CCRBK63 ZmRPAMSH-1 P0026 C5 CGEUZ26 ZmRPAMSH-2 P0002 TBDCGEVJ74 ZmRPAMSH-3 P0002 TBD CHSBX01 ZmRPABMS-4 P0118 C5 CIMME04ZmRPAMSH-5 P0114 C5 CRTBB78 ZmRPAMSH-6 P0041 C5 CVRAP89 ZmRPAMSH-7 P0057C5

Example 2 Transformation and Regeneration of Transgenic Plants

[0150] Immature maize embryos from greenhouse donor plants are bombardedwith a plasmid containing the RPA antisense sequence of the inventionoperably linked to a pathogen-inducible promoter (FIG. 2) plus a plasmidcontaining the selectable marker gene PAT (Wohlleben et al. (1988) Gene70:25-37) that confers resistance to the herbicide Bialaphos.Transformation is performed as follows. All media recipes are in theAppendix.

[0151] Preparation of Target Tissue

[0152] The ears are surface sterilized in 30% Chlorox bleach plus 0.5%Micro detergent for 20 minutes, and rinsed two times with sterile water.The immature embryos are excised and placed embryo axis side down(scutellum side up), 25 embryos per plate, on 560Y medium for 4 hoursand then aligned within the 2.5-cm target zone in preparation forbombardment.

[0153] Preparation of DNA

[0154] A plasmid vector comprising the RPA sequence of the inventionoperably linked to a ubiquitin promoter is made. This plasmid DNA plusplasmid DNA containing a PAT selectable marker is precipitated onto 1.1μm (average diameter) tungsten pellets using a CaCl₂ precipitationprocedure as follows:

[0155] 100 μl prepared tungsten particles in water

[0156] 10 μl (1 μg) DNA in TrisEDTA buffer (1 μg total)

[0157] 100 μl 2.5 M CaC1₂

[0158] 10 μl 0.1 M spermidine

[0159] Each reagent is added sequentially to the tungsten particlesuspension, while maintained on the multitube vortexer. The finalmixture is sonicated briefly and allowed to incubate under constantvortexing for 10 minutes. After the precipitation period, the tubes arecentrifuged briefly, liquid removed, washed with 500 ml 100% ethanol,and centrifuged for 30 seconds. Again the liquid is removed, and 105 μl100% ethanol is added to the final tungsten particle pellet. Forparticle gun bombardment, the tungsten/DNA particles are brieflysonicated and 10 μl spotted onto the center of each macrocarrier andallowed to dry about 2 minutes before bombardment.

[0160] Particle Gun Treatment

[0161] The sample plates are bombarded at level #4 in particle gun#HE34-1 or #HE34-2. All samples receive a single shot at 650 PSI, with atotal of ten aliquots taken from each tube of prepared particles/DNA.

[0162] Subsequent Treatment

[0163] Following bombardment, the embryos are kept on 560Y medium for 2days, then transferred to 560R selection medium containing 3 mg/literBialaphos, and subcultured every 2 weeks. After approximately 10 weeksof selection, selection-resistant callus clones are transferred to 288Jmedium to initiate plant regeneration. Following somatic embryomaturation (2-4 weeks), well-developed somatic embryos are transferredto medium for germination and transferred to the lighted culture room.Approximately 7-10 days later, developing plantlets are transferred to272V hormone-free medium in tubes for 7-10 days until plantlets are wellestablished. Plants are then transferred to inserts in flats (equivalentto 2.5″ pot) containing pofting soil and grown for 1 week in a growthchamber, subsequently grown an additional 1-2 weeks in the greenhouse,then transferred to classic 600 pots (1.6 gallon) and grown to maturity.Plants are monitored and scored for expression of the RPA gene ofinterest. Ingredient Amount Unit D-I H₂O 950.000 MI MS Salts (GIBCO11117-074) 4.300 G Myo-Inositol 0.100 G MS Vitamins Stock Solution ##5.000 MI Sucrose 40.000 G Bacto-Agar @ 6.000 G

[0164] Ingredient Amount Unit D-I H₂O 950.000 MI MS Salts 4.300 GMyo-Inositol 0.100 G MS Vitamins Stock Solution ## 5.000 MI Zeatin .5mg/ml 1.000 MI Sucrose 60.000 G Gelrite @ 3.000 G Indoleacetic Acid 0.5mg/ml # 2.000 MI 0.1 mM Abscisic Acid 1.000 MI Bialaphos 1 mg/ml # 3.000MI

[0165] Ingredient Amount Unit D-I Water, Filtered 950.000 MI CHU (N6)Basal Salts (SIGMA C-1416) 4.000 G Eriksson's Vitamin Mix (1000XSIGMA-1511 1.000 MI Thiamine · HCL 0.4 mg/ml 1.250 MI Sucrose 30.000 G2, 4-D 0.5 mg/ml 4.000 MI Gelrite @ 3.000 G Silver Nitrate 2 mg/ml #0.425 MI Bialaphos 1 mg/ml # 3.000 MI

[0166] Ingredient Amount Unit D-I Water, Filtered 950.000 MI CHU (N6)Basal Salts (SIGMA C-1416) 4.000 G Eriksson's Vitamin Mix (1000XSIGMA-1511 1.000 MI Thiamine · HCL 0.4 mg/ml 1.250 MI Sucrose 120.000 G2,4-D 0.5 mg/ml 2.000 MI L-Proline 2.880 G Gelrite @ 2.000 G SilverNitrate 2 mg/ml # 4.250 MI

[0167] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1 22 1 2497 DNA Zea Mays CDS (157)...(2025) Coding sequence for theMaize RPA Large Subunit Homologue-1 1 ccttatcata ttataagcgc gcgtagccttggcagctcga cgcatcttcg cctccgctca 60 acgctcgccc acgcccccag cccccaccgatccacgagaa accttctcgc ctccgcggga 120 cgattcgcca gggagagcaa aggtagcagaggcgcc atg gac gct gcc aag tcg 174 Met Asp Ala Ala Lys Ser 1 5 gtg acgccg ggc gcc gtg tcc tac atc ctg gcg cac ccg tct acg ggc 222 Val Thr ProGly Ala Val Ser Tyr Ile Leu Ala His Pro Ser Thr Gly 10 15 20 tcc gat ggcgcc gtg tcg gat ctc gtc gtt cag gtc ctc gat ctc aag 270 Ser Asp Gly AlaVal Ser Asp Leu Val Val Gln Val Leu Asp Leu Lys 25 30 35 tcc atc ggc atgggc agc cgg ttc agt ttc acg gca tcc gat ggg aac 318 Ser Ile Gly Met GlySer Arg Phe Ser Phe Thr Ala Ser Asp Gly Asn 40 45 50 gac aaa atc aag gcgatg ctc ccc act tac ttt gcg tcg gag gtc cac 366 Asp Lys Ile Lys Ala MetLeu Pro Thr Tyr Phe Ala Ser Glu Val His 55 60 65 70 tcc ggc aat ctg aagaat ttc ggt ctc atc cgc atc ctc gac tac act 414 Ser Gly Asn Leu Lys AsnPhe Gly Leu Ile Arg Ile Leu Asp Tyr Thr 75 80 85 tgc aac tcc gtc aaa ggcaac gct gac aaa gtc ctg att gtc gtc aaa 462 Cys Asn Ser Val Lys Gly AsnAla Asp Lys Val Leu Ile Val Val Lys 90 95 100 tgc gag act gtg tgc gaagcg ctc gac gcc gag atc aac ggc gag gcc 510 Cys Glu Thr Val Cys Glu AlaLeu Asp Ala Glu Ile Asn Gly Glu Ala 105 110 115 aag aaa gag gat cct ccaatt gtg ctg aag cct aaa gac gaa ggc tca 558 Lys Lys Glu Asp Pro Pro IleVal Leu Lys Pro Lys Asp Glu Gly Ser 120 125 130 gtc gtg gct gag gaa acaaat tct ccc cca ctc gtg atg aag cct aag 606 Val Val Ala Glu Glu Thr AsnSer Pro Pro Leu Val Met Lys Pro Lys 135 140 145 150 caa gag gtg aag tccgcg tcc cag atc gtg act gag cag cgt gga aat 654 Gln Glu Val Lys Ser AlaSer Gln Ile Val Thr Glu Gln Arg Gly Asn 155 160 165 gct gct cct gcc acgcgc ctt tcc atg aca agg agg gtc cat ccc ttg 702 Ala Ala Pro Ala Thr ArgLeu Ser Met Thr Arg Arg Val His Pro Leu 170 175 180 atc act ctg aac ccctac cag ggt aac tgg gtc att aag gtg cgg gtc 750 Ile Thr Leu Asn Pro TyrGln Gly Asn Trp Val Ile Lys Val Arg Val 185 190 195 acg agc aaa ggc aatctg aga acc tac agg aat gct cgt gga gaa ggc 798 Thr Ser Lys Gly Asn LeuArg Thr Tyr Arg Asn Ala Arg Gly Glu Gly 200 205 210 tgc gtc ttc aac gtagag ctt act gat gag gat ggc acc cag atc cag 846 Cys Val Phe Asn Val GluLeu Thr Asp Glu Asp Gly Thr Gln Ile Gln 215 220 225 230 gcc acc atg tttaac gag gct gca aag aag ttc tat cca att ttt gag 894 Ala Thr Met Phe AsnGlu Ala Ala Lys Lys Phe Tyr Pro Ile Phe Glu 235 240 245 ctg gga aag gtctat tat gtc tca aaa gga tct ctt aga att gcc aac 942 Leu Gly Lys Val TyrTyr Val Ser Lys Gly Ser Leu Arg Ile Ala Asn 250 255 260 aag cag ttc aagaca gtc aaa aat gac tat gag ttg tca cta aac gag 990 Lys Gln Phe Lys ThrVal Lys Asn Asp Tyr Glu Leu Ser Leu Asn Glu 265 270 275 aat gct att gttgaa gaa gca gag ggg gag act ttc ctt cca cca gtg 1038 Asn Ala Ile Val GluGlu Ala Glu Gly Glu Thr Phe Leu Pro Pro Val 280 285 290 caa tac aac cttgtc aag att gat cag cta gga cca tac gtc ggt ggc 1086 Gln Tyr Asn Leu ValLys Ile Asp Gln Leu Gly Pro Tyr Val Gly Gly 295 300 305 310 agg gag cttgta gat att gtt ggt gtg gtt cag agc gta tct ccc aca 1134 Arg Glu Leu ValAsp Ile Val Gly Val Val Gln Ser Val Ser Pro Thr 315 320 325 ctc agt gttagg aga aag att gac aac gag aca ata ccg aag cgt gac 1182 Leu Ser Val ArgArg Lys Ile Asp Asn Glu Thr Ile Pro Lys Arg Asp 330 335 340 att gtt gtagca gac gac tct ggc aaa act gtt act att tct ctc tgg 1230 Ile Val Val AlaAsp Asp Ser Gly Lys Thr Val Thr Ile Ser Leu Trp 345 350 355 aat gat cttgct act acg act ggc caa gag ctt ttg gac atg gtt gac 1278 Asn Asp Leu AlaThr Thr Thr Gly Gln Glu Leu Leu Asp Met Val Asp 360 365 370 agt tcg cctgtt gtt gcg ata aag agc cta aaa gta tct gac ttc caa 1326 Ser Ser Pro ValVal Ala Ile Lys Ser Leu Lys Val Ser Asp Phe Gln 375 380 385 390 ggc gtgtct ctt tca act att ggc aga agt act ctc gag att aat cct 1374 Gly Val SerLeu Ser Thr Ile Gly Arg Ser Thr Leu Glu Ile Asn Pro 395 400 405 gac ctgcct gag gct aag aat ctt aag tcc tgg tat gac tct gaa ggc 1422 Asp Leu ProGlu Ala Lys Asn Leu Lys Ser Trp Tyr Asp Ser Glu Gly 410 415 420 aaa gatact tca ctg gca cca atc agt gca gaa gcg ggt gcc aca cgc 1470 Lys Asp ThrSer Leu Ala Pro Ile Ser Ala Glu Ala Gly Ala Thr Arg 425 430 435 gct ggtggt ttc aag tcc atg tat tct gat aga gtt ttt ctg tct cac 1518 Ala Gly GlyPhe Lys Ser Met Tyr Ser Asp Arg Val Phe Leu Ser His 440 445 450 atc accagt gat cct gct atg ggc cag gaa aag cct gtt ttc ttc agt 1566 Ile Thr SerAsp Pro Ala Met Gly Gln Glu Lys Pro Val Phe Phe Ser 455 460 465 470 ctgtac gcc atc ata agc cac atc aag cct gat cag aat atg tgg tac 1614 Leu TyrAla Ile Ile Ser His Ile Lys Pro Asp Gln Asn Met Trp Tyr 475 480 485 cgtgct tgc acg acc tgt aac aag aag gtg act gaa gct ttt ggg tct 1662 Arg AlaCys Thr Thr Cys Asn Lys Lys Val Thr Glu Ala Phe Gly Ser 490 495 500 ggatac tgg tgc gag ggg tgc caa aag aat gac tct gag tgc tcg ctg 1710 Gly TyrTrp Cys Glu Gly Cys Gln Lys Asn Asp Ser Glu Cys Ser Leu 505 510 515 aggtac atc atg gtg atc aag ctc tcc gat ccc act ggt gag gct tgg 1758 Arg TyrIle Met Val Ile Lys Leu Ser Asp Pro Thr Gly Glu Ala Trp 520 525 530 gtgtcc gtg ttc aac gag cat gcg gag aag atc att ggc tgc agc gcc 1806 Val SerVal Phe Asn Glu His Ala Glu Lys Ile Ile Gly Cys Ser Ala 535 540 545 550gac gag ctt gat cgg atc agg aaa gag gag ggg gac gac agc tac gtt 1854 AspGlu Leu Asp Arg Ile Arg Lys Glu Glu Gly Asp Asp Ser Tyr Val 555 560 565ctc aag ctc aag gaa gcc acc tgg gtt cct cac ctg ttc cgc gtc agc 1902 LeuLys Leu Lys Glu Ala Thr Trp Val Pro His Leu Phe Arg Val Ser 570 575 580gtc aca cag cat gaa tac atg aac gag aag agg cag aga atc acc gtg 1950 ValThr Gln His Glu Tyr Met Asn Glu Lys Arg Gln Arg Ile Thr Val 585 590 595agg ggt gaa gca ccg gtc gac ttc gca gct gag tcc aag tac ttg ctt 1998 ArgGly Glu Ala Pro Val Asp Phe Ala Ala Glu Ser Lys Tyr Leu Leu 600 605 610gaa gag atc gcg aag ctc acc gct tgc tagaagacgc agtctttctg 2045 Glu GluIle Ala Lys Leu Thr Ala Cys 615 620 gtggttcttg aaggactggc ccccgatatgtctccctctc agtttttctt ttgagctcca 2105 gtaacttgat tactgttctg tgtgttgctctcactgggtt ttagcacttc tgtaaggtat 2165 atgtagatgc tagtttacct tggtgtcaaggaacagatgc tattataagc cttgcaaaat 2225 tgcagttcca attccgtgta tctgcaaccttgagcaaata gggaaagatt atgagtacta 2285 attgatgatg ttaggtcgct gcagctaacaagtgtttggt ttttagtgac tactgtttag 2345 tccctatatt ttattctatt ttagtatttaaggttgcgtt tggttgcgtc gactagacat 2405 gttgtgcgtg tccgatgagt ctattattgaagcacaaaat tgggaataaa aaaaaaaaaa 2465 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaa 2497 2 623 PRT Zea Mays 2 Met Asp Ala Ala Lys Ser Val Thr Pro Gly AlaVal Ser Tyr Ile Leu 1 5 10 15 Ala His Pro Ser Thr Gly Ser Asp Gly AlaVal Ser Asp Leu Val Val 20 25 30 Gln Val Leu Asp Leu Lys Ser Ile Gly MetGly Ser Arg Phe Ser Phe 35 40 45 Thr Ala Ser Asp Gly Asn Asp Lys Ile LysAla Met Leu Pro Thr Tyr 50 55 60 Phe Ala Ser Glu Val His Ser Gly Asn LeuLys Asn Phe Gly Leu Ile 65 70 75 80 Arg Ile Leu Asp Tyr Thr Cys Asn SerVal Lys Gly Asn Ala Asp Lys 85 90 95 Val Leu Ile Val Val Lys Cys Glu ThrVal Cys Glu Ala Leu Asp Ala 100 105 110 Glu Ile Asn Gly Glu Ala Lys LysGlu Asp Pro Pro Ile Val Leu Lys 115 120 125 Pro Lys Asp Glu Gly Ser ValVal Ala Glu Glu Thr Asn Ser Pro Pro 130 135 140 Leu Val Met Lys Pro LysGln Glu Val Lys Ser Ala Ser Gln Ile Val 145 150 155 160 Thr Glu Gln ArgGly Asn Ala Ala Pro Ala Thr Arg Leu Ser Met Thr 165 170 175 Arg Arg ValHis Pro Leu Ile Thr Leu Asn Pro Tyr Gln Gly Asn Trp 180 185 190 Val IleLys Val Arg Val Thr Ser Lys Gly Asn Leu Arg Thr Tyr Arg 195 200 205 AsnAla Arg Gly Glu Gly Cys Val Phe Asn Val Glu Leu Thr Asp Glu 210 215 220Asp Gly Thr Gln Ile Gln Ala Thr Met Phe Asn Glu Ala Ala Lys Lys 225 230235 240 Phe Tyr Pro Ile Phe Glu Leu Gly Lys Val Tyr Tyr Val Ser Lys Gly245 250 255 Ser Leu Arg Ile Ala Asn Lys Gln Phe Lys Thr Val Lys Asn AspTyr 260 265 270 Glu Leu Ser Leu Asn Glu Asn Ala Ile Val Glu Glu Ala GluGly Glu 275 280 285 Thr Phe Leu Pro Pro Val Gln Tyr Asn Leu Val Lys IleAsp Gln Leu 290 295 300 Gly Pro Tyr Val Gly Gly Arg Glu Leu Val Asp IleVal Gly Val Val 305 310 315 320 Gln Ser Val Ser Pro Thr Leu Ser Val ArgArg Lys Ile Asp Asn Glu 325 330 335 Thr Ile Pro Lys Arg Asp Ile Val ValAla Asp Asp Ser Gly Lys Thr 340 345 350 Val Thr Ile Ser Leu Trp Asn AspLeu Ala Thr Thr Thr Gly Gln Glu 355 360 365 Leu Leu Asp Met Val Asp SerSer Pro Val Val Ala Ile Lys Ser Leu 370 375 380 Lys Val Ser Asp Phe GlnGly Val Ser Leu Ser Thr Ile Gly Arg Ser 385 390 395 400 Thr Leu Glu IleAsn Pro Asp Leu Pro Glu Ala Lys Asn Leu Lys Ser 405 410 415 Trp Tyr AspSer Glu Gly Lys Asp Thr Ser Leu Ala Pro Ile Ser Ala 420 425 430 Glu AlaGly Ala Thr Arg Ala Gly Gly Phe Lys Ser Met Tyr Ser Asp 435 440 445 ArgVal Phe Leu Ser His Ile Thr Ser Asp Pro Ala Met Gly Gln Glu 450 455 460Lys Pro Val Phe Phe Ser Leu Tyr Ala Ile Ile Ser His Ile Lys Pro 465 470475 480 Asp Gln Asn Met Trp Tyr Arg Ala Cys Thr Thr Cys Asn Lys Lys Val485 490 495 Thr Glu Ala Phe Gly Ser Gly Tyr Trp Cys Glu Gly Cys Gln LysAsn 500 505 510 Asp Ser Glu Cys Ser Leu Arg Tyr Ile Met Val Ile Lys LeuSer Asp 515 520 525 Pro Thr Gly Glu Ala Trp Val Ser Val Phe Asn Glu HisAla Glu Lys 530 535 540 Ile Ile Gly Cys Ser Ala Asp Glu Leu Asp Arg IleArg Lys Glu Glu 545 550 555 560 Gly Asp Asp Ser Tyr Val Leu Lys Leu LysGlu Ala Thr Trp Val Pro 565 570 575 His Leu Phe Arg Val Ser Val Thr GlnHis Glu Tyr Met Asn Glu Lys 580 585 590 Arg Gln Arg Ile Thr Val Arg GlyGlu Ala Pro Val Asp Phe Ala Ala 595 600 605 Glu Ser Lys Tyr Leu Leu GluGlu Ile Ala Lys Leu Thr Ala Cys 610 615 620 3 2202 DNA Zea Mays CDS(91)...(1941) Coding Region for Maize RPA Large Subunit Homologue-2 3acgttccccc cacgccccaa cctatccacg cgaaaccttc tttcccccgg gagacgattc 60gtcagggaga ggaaagaggc aagaggggcc atg gac gct gcc aag ttg gtg acg 114 MetAsp Ala Ala Lys Leu Val Thr 1 5 ccg gtc gct gtg tct cac att ctg gcg cacccg tcg gcg ggc tcc gac 162 Pro Val Ala Val Ser His Ile Leu Ala His ProSer Ala Gly Ser Asp 10 15 20 ggc gca gtg acc gat ctc gtc gtt cag gtc ctcgac ctg aag tcc gtc 210 Gly Ala Val Thr Asp Leu Val Val Gln Val Leu AspLeu Lys Ser Val 25 30 35 40 ggc acg ggc agc cgg ttc agt ttc aca gca actgac ggg aag gat aag 258 Gly Thr Gly Ser Arg Phe Ser Phe Thr Ala Thr AspGly Lys Asp Lys 45 50 55 atc aag gcg atg ctt ccc acc aac ttc ggg tcg gaggtc cgc tct ggc 306 Ile Lys Ala Met Leu Pro Thr Asn Phe Gly Ser Glu ValArg Ser Gly 60 65 70 aac ctg aag aac ctc ggc ctc atc cgc atc atc gac tacact tgc aac 354 Asn Leu Lys Asn Leu Gly Leu Ile Arg Ile Ile Asp Tyr ThrCys Asn 75 80 85 gtc gtc aaa ggc aaa gat gac aaa gtc ttg gtt gtc atc aaatgc gag 402 Val Val Lys Gly Lys Asp Asp Lys Val Leu Val Val Ile Lys CysGlu 90 95 100 ctt gtg tgc caa gcg ctt gac gcc gag atc aac ggc gag gccaaa aaa 450 Leu Val Cys Gln Ala Leu Asp Ala Glu Ile Asn Gly Glu Ala LysLys 105 110 115 120 gag gag cct cca att gtg ctg aag cct aag gac gaa tgcgtg ggc gtg 498 Glu Glu Pro Pro Ile Val Leu Lys Pro Lys Asp Glu Cys ValGly Val 125 130 135 act tcc cca ctc gct atg aag ccc aag cag gag gtg aagtct gcg tcc 546 Thr Ser Pro Leu Ala Met Lys Pro Lys Gln Glu Val Lys SerAla Ser 140 145 150 cag atc gtg aat gag cag cgt gga aat act gct cct gtcaag ccc ctt 594 Gln Ile Val Asn Glu Gln Arg Gly Asn Thr Ala Pro Val LysPro Leu 155 160 165 tcc atg aca aag agg gtc cat cct ttg atc act ctg aacccc tac cag 642 Ser Met Thr Lys Arg Val His Pro Leu Ile Thr Leu Asn ProTyr Gln 170 175 180 ggt aac tgg gtc att aag gtg cgg gtc acg agc aaa ggcaac ctg aga 690 Gly Asn Trp Val Ile Lys Val Arg Val Thr Ser Lys Gly AsnLeu Arg 185 190 195 200 acc tac agg aat gct cgc gga gaa ggc tgt gtc ttcaat gta gag ctc 738 Thr Tyr Arg Asn Ala Arg Gly Glu Gly Cys Val Phe AsnVal Glu Leu 205 210 215 acc gat gag gat ggc acc cag atc caa gcc acc atgttt aat gac gct 786 Thr Asp Glu Asp Gly Thr Gln Ile Gln Ala Thr Met PheAsn Asp Ala 220 225 230 gca aag aag ttc tat ccg att ttt gag ctg gga aaggtc tat tat gtc 834 Ala Lys Lys Phe Tyr Pro Ile Phe Glu Leu Gly Lys ValTyr Tyr Val 235 240 245 tca aaa gga tct ctt aga att gct aac aag cag ttcaag act gtc caa 882 Ser Lys Gly Ser Leu Arg Ile Ala Asn Lys Gln Phe LysThr Val Gln 250 255 260 aat gac tac gag atg tca cta aac gag aat gct attgtt gaa gaa gca 930 Asn Asp Tyr Glu Met Ser Leu Asn Glu Asn Ala Ile ValGlu Glu Ala 265 270 275 280 gag ggg gag act tgc att ccg caa gtg caa tacaac ctt gtc aag att 978 Glu Gly Glu Thr Cys Ile Pro Gln Val Gln Tyr AsnLeu Val Lys Ile 285 290 295 gat caa cta gga tca tat gtc ggt ggc agg gaactt gta gat att gtt 1026 Asp Gln Leu Gly Ser Tyr Val Gly Gly Arg Glu LeuVal Asp Ile Val 300 305 310 ggt gtg gtt cag agc gta tct ccc aca ctc agtgtc agg aga aag att 1074 Gly Val Val Gln Ser Val Ser Pro Thr Leu Ser ValArg Arg Lys Ile 315 320 325 gac aac gag aca ata ccg aag cgt gac att gttgtg gcg gat gac tct 1122 Asp Asn Glu Thr Ile Pro Lys Arg Asp Ile Val ValAla Asp Asp Ser 330 335 340 ggc aaa act gtt agt atc tct ctt tgg aat gatctt gct act acg act 1170 Gly Lys Thr Val Ser Ile Ser Leu Trp Asn Asp LeuAla Thr Thr Thr 345 350 355 360 ggg caa gag ctt ttg gac atg gct gac agttcg cct gtt gtt gcg ata 1218 Gly Gln Glu Leu Leu Asp Met Ala Asp Ser SerPro Val Val Ala Ile 365 370 375 aag agc cta aaa gtg tct gac ttt caa ggcgtg tct ctt tct act gta 1266 Lys Ser Leu Lys Val Ser Asp Phe Gln Gly ValSer Leu Ser Thr Val 380 385 390 ggc aaa agt act ctt gcg att aat cct gatcta cac gag gct cag aat 1314 Gly Lys Ser Thr Leu Ala Ile Asn Pro Asp LeuHis Glu Ala Gln Asn 395 400 405 ctc aag tca tgg tat gac tct gaa ggc aaagat act tcg ctg gca cca 1362 Leu Lys Ser Trp Tyr Asp Ser Glu Gly Lys AspThr Ser Leu Ala Pro 410 415 420 att ggt gca gaa atg ggt gcc gca cgg gccggt ggc ttc aag tcc acg 1410 Ile Gly Ala Glu Met Gly Ala Ala Arg Ala GlyGly Phe Lys Ser Thr 425 430 435 440 tat tct gat aga gtt ttt ctg tct cacatt act agt gat cct gcc atg 1458 Tyr Ser Asp Arg Val Phe Leu Ser His IleThr Ser Asp Pro Ala Met 445 450 455 ggc cag gaa aag cct gtt ttc ttc agtttg tat gcc acc ata agc cac 1506 Gly Gln Glu Lys Pro Val Phe Phe Ser LeuTyr Ala Thr Ile Ser His 460 465 470 atc aag cct gac cag aac atg tgg taccgt gct tgc aag acc tgc aac 1554 Ile Lys Pro Asp Gln Asn Met Trp Tyr ArgAla Cys Lys Thr Cys Asn 475 480 485 aag aag gtg act gaa act ttt gga tctgga tac tgg tgc gag gga tgc 1602 Lys Lys Val Thr Glu Thr Phe Gly Ser GlyTyr Trp Cys Glu Gly Cys 490 495 500 caa aag aat gac tcg gaa tgc tca ctgaga tac atc atg gtc atc aag 1650 Gln Lys Asn Asp Ser Glu Cys Ser Leu ArgTyr Ile Met Val Ile Lys 505 510 515 520 gtc tcc gat cct act ggc gag gcatgg ttc tct gtg ttc aac gag cat 1698 Val Ser Asp Pro Thr Gly Glu Ala TrpPhe Ser Val Phe Asn Glu His 525 530 535 gca gag aag atc att ggc tgc agcgcc gac gag ctt gat cgg atc agg 1746 Ala Glu Lys Ile Ile Gly Cys Ser AlaAsp Glu Leu Asp Arg Ile Arg 540 545 550 aaa gag gag ggg gac gac agt tatgtt ctg aag ctc aag gaa gcc acc 1794 Lys Glu Glu Gly Asp Asp Ser Tyr ValLeu Lys Leu Lys Glu Ala Thr 555 560 565 tgg gtt cct cac ctg ttc cgc gtcagc gtc aca cag cat gaa tac aat 1842 Trp Val Pro His Leu Phe Arg Val SerVal Thr Gln His Glu Tyr Asn 570 575 580 aac gag aaa agg cag aga atc actgtg agg agt gaa gcg ccg gtc gag 1890 Asn Glu Lys Arg Gln Arg Ile Thr ValArg Ser Glu Ala Pro Val Glu 585 590 595 600 cac gca gct gaa tcc aag tacctg ctt gaa cag ata gcg aag ctt act 1938 His Ala Ala Glu Ser Lys Tyr LeuLeu Glu Gln Ile Ala Lys Leu Thr 605 610 615 gct tgatagtaga agatgcaaccttactgcaaa tagcgaggat tattaggact 1991 Ala aattgatggt gtcaggtcattgcggcccta agctttagct ctctatcagc agtcagatgt 2051 attaaccatt ccctgctctaatagtcatct atcagcagtc agatgtattt aaccaaaaaa 2111 aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaagggcgg ccgctctaga 2171 ggatccaagc ttacgtacgcgtgcatgcga c 2202 4 617 PRT Zea Mays 4 Met Asp Ala Ala Lys Leu Val ThrPro Val Ala Val Ser His Ile Leu 1 5 10 15 Ala His Pro Ser Ala Gly SerAsp Gly Ala Val Thr Asp Leu Val Val 20 25 30 Gln Val Leu Asp Leu Lys SerVal Gly Thr Gly Ser Arg Phe Ser Phe 35 40 45 Thr Ala Thr Asp Gly Lys AspLys Ile Lys Ala Met Leu Pro Thr Asn 50 55 60 Phe Gly Ser Glu Val Arg SerGly Asn Leu Lys Asn Leu Gly Leu Ile 65 70 75 80 Arg Ile Ile Asp Tyr ThrCys Asn Val Val Lys Gly Lys Asp Asp Lys 85 90 95 Val Leu Val Val Ile LysCys Glu Leu Val Cys Gln Ala Leu Asp Ala 100 105 110 Glu Ile Asn Gly GluAla Lys Lys Glu Glu Pro Pro Ile Val Leu Lys 115 120 125 Pro Lys Asp GluCys Val Gly Val Thr Ser Pro Leu Ala Met Lys Pro 130 135 140 Lys Gln GluVal Lys Ser Ala Ser Gln Ile Val Asn Glu Gln Arg Gly 145 150 155 160 AsnThr Ala Pro Val Lys Pro Leu Ser Met Thr Lys Arg Val His Pro 165 170 175Leu Ile Thr Leu Asn Pro Tyr Gln Gly Asn Trp Val Ile Lys Val Arg 180 185190 Val Thr Ser Lys Gly Asn Leu Arg Thr Tyr Arg Asn Ala Arg Gly Glu 195200 205 Gly Cys Val Phe Asn Val Glu Leu Thr Asp Glu Asp Gly Thr Gln Ile210 215 220 Gln Ala Thr Met Phe Asn Asp Ala Ala Lys Lys Phe Tyr Pro IlePhe 225 230 235 240 Glu Leu Gly Lys Val Tyr Tyr Val Ser Lys Gly Ser LeuArg Ile Ala 245 250 255 Asn Lys Gln Phe Lys Thr Val Gln Asn Asp Tyr GluMet Ser Leu Asn 260 265 270 Glu Asn Ala Ile Val Glu Glu Ala Glu Gly GluThr Cys Ile Pro Gln 275 280 285 Val Gln Tyr Asn Leu Val Lys Ile Asp GlnLeu Gly Ser Tyr Val Gly 290 295 300 Gly Arg Glu Leu Val Asp Ile Val GlyVal Val Gln Ser Val Ser Pro 305 310 315 320 Thr Leu Ser Val Arg Arg LysIle Asp Asn Glu Thr Ile Pro Lys Arg 325 330 335 Asp Ile Val Val Ala AspAsp Ser Gly Lys Thr Val Ser Ile Ser Leu 340 345 350 Trp Asn Asp Leu AlaThr Thr Thr Gly Gln Glu Leu Leu Asp Met Ala 355 360 365 Asp Ser Ser ProVal Val Ala Ile Lys Ser Leu Lys Val Ser Asp Phe 370 375 380 Gln Gly ValSer Leu Ser Thr Val Gly Lys Ser Thr Leu Ala Ile Asn 385 390 395 400 ProAsp Leu His Glu Ala Gln Asn Leu Lys Ser Trp Tyr Asp Ser Glu 405 410 415Gly Lys Asp Thr Ser Leu Ala Pro Ile Gly Ala Glu Met Gly Ala Ala 420 425430 Arg Ala Gly Gly Phe Lys Ser Thr Tyr Ser Asp Arg Val Phe Leu Ser 435440 445 His Ile Thr Ser Asp Pro Ala Met Gly Gln Glu Lys Pro Val Phe Phe450 455 460 Ser Leu Tyr Ala Thr Ile Ser His Ile Lys Pro Asp Gln Asn MetTrp 465 470 475 480 Tyr Arg Ala Cys Lys Thr Cys Asn Lys Lys Val Thr GluThr Phe Gly 485 490 495 Ser Gly Tyr Trp Cys Glu Gly Cys Gln Lys Asn AspSer Glu Cys Ser 500 505 510 Leu Arg Tyr Ile Met Val Ile Lys Val Ser AspPro Thr Gly Glu Ala 515 520 525 Trp Phe Ser Val Phe Asn Glu His Ala GluLys Ile Ile Gly Cys Ser 530 535 540 Ala Asp Glu Leu Asp Arg Ile Arg LysGlu Glu Gly Asp Asp Ser Tyr 545 550 555 560 Val Leu Lys Leu Lys Glu AlaThr Trp Val Pro His Leu Phe Arg Val 565 570 575 Ser Val Thr Gln His GluTyr Asn Asn Glu Lys Arg Gln Arg Ile Thr 580 585 590 Val Arg Ser Glu AlaPro Val Glu His Ala Ala Glu Ser Lys Tyr Leu 595 600 605 Leu Glu Gln IleAla Lys Leu Thr Ala 610 615 5 630 PRT Oryza sativa 5 Met Asp Ser Asp AlaAla Pro Ser Val Thr Pro Gly Ala Val Ala Phe 1 5 10 15 Val Leu Glu AsnAla Ser Pro Asp Ala Ala Thr Gly Val Pro Val Pro 20 25 30 Glu Ile Val LeuGln Val Val Asp Leu Lys Pro Ile Gly Thr Arg Phe 35 40 45 Thr Phe Leu AlaSer Asp Gly Lys Asp Lys Ile Lys Thr Met Leu Leu 50 55 60 Thr Gln Leu AlaPro Glu Val Arg Ser Gly Asn Ile Gln Asn Leu Gly 65 70 75 80 Val Ile ArgVal Leu Asp Tyr Thr Cys Asn Thr Ile Gly Glu Lys Gln 85 90 95 Glu Lys ValLeu Ile Ile Thr Lys Leu Glu Val Val Phe Lys Ala Leu 100 105 110 Asp SerGlu Ile Lys Cys Glu Ala Glu Lys Gln Glu Glu Lys Pro Ala 115 120 125 IleLeu Leu Ser Pro Lys Glu Glu Ser Val Val Leu Ser Lys Pro Thr 130 135 140Asn Ala Pro Pro Leu Pro Pro Val Val Leu Lys Pro Lys Gln Glu Val 145 150155 160 Lys Ser Ala Ser Gln Ile Val Asn Glu Gln Arg Gly Asn Ala Ala Pro165 170 175 Ala Ala Arg Leu Ala Met Thr Arg Arg Val His Pro Leu Ile SerLeu 180 185 190 Asn Pro Tyr Gln Gly Asn Trp Ile Ile Lys Val Arg Val ThrSer Lys 195 200 205 Gly Asn Leu Arg Thr Tyr Lys Asn Ala Arg Gly Glu GlyCys Val Phe 210 215 220 Asn Val Glu Leu Thr Asp Val Asp Gly Thr Gln IleGln Ala Thr Met 225 230 235 240 Phe Asn Glu Ala Ala Lys Lys Phe Tyr ProMet Phe Glu Leu Gly Lys 245 250 255 Val Tyr Tyr Ile Ser Lys Gly Ser LeuArg Val Ala Asn Lys Gln Phe 260 265 270 Lys Thr Val His Asn Asp Tyr GluMet Thr Leu Asn Glu Asn Ala Val 275 280 285 Val Glu Glu Ala Glu Gly GluThr Phe Ile Pro Gln Ile Gln Tyr Asn 290 295 300 Phe Val Lys Ile Asp GlnLeu Gly Pro Tyr Val Gly Gly Arg Glu Leu 305 310 315 320 Val Asp Val IleGly Val Val Gln Ser Val Ser Pro Thr Leu Ser Val 325 330 335 Arg Arg LysIle Asp Asn Glu Thr Ile Pro Lys Arg Asp Ile Val Val 340 345 350 Ala AspAsp Ser Ser Lys Thr Val Thr Ile Ser Leu Trp Asn Asp Leu 355 360 365 AlaThr Thr Thr Gly Gln Glu Leu Leu Asp Met Val Asp Ser Ala Pro 370 375 380Ile Ile Ala Ile Lys Ser Leu Lys Val Ser Asp Phe Gln Gly Leu Ser 385 390395 400 Leu Ser Thr Val Gly Arg Ser Thr Ile Val Val Asn Pro Asp Leu Pro405 410 415 Glu Ala Glu Gln Leu Arg Ala Trp Tyr Asp Ser Glu Gly Lys GlyThr 420 425 430 Ser Met Ala Ser Ile Gly Ser Asp Met Gly Ala Ser Arg ValGly Gly 435 440 445 Ala Arg Ser Met Tyr Ser Asp Arg Val Phe Leu Ser HisIle Thr Ser 450 455 460 Asp Pro Asn Leu Gly Gln Asp Lys Pro Val Phe PheSer Leu Asn Ala 465 470 475 480 Tyr Ile Ser Leu Ile Lys Pro Asp Gln ThrMet Trp Tyr Arg Ala Cys 485 490 495 Lys Thr Cys Asn Lys Lys Val Thr GluAla Met Gly Ser Gly Tyr Trp 500 505 510 Cys Glu Gly Cys Gln Lys Asn AspAla Glu Cys Ser Leu Arg Tyr Ile 515 520 525 Met Val Ile Lys Val Ser AspPro Thr Gly Glu Ala Trp Leu Ser Leu 530 535 540 Phe Asn Asp Gln Ala GluArg Ile Val Gly Cys Ser Ala Asp Glu Leu 545 550 555 560 Asp Arg Ile ArgLys Glu Glu Gly Asp Asp Ser Tyr Leu Leu Lys Leu 565 570 575 Lys Glu AlaThr Trp Val Pro His Leu Phe Arg Val Ser Val Thr Gln 580 585 590 Asn GluTyr Met Asn Glu Lys Arg Gln Arg Ile Thr Val Arg Ser Glu 595 600 605 AlaPro Val Asp His Ala Ala Glu Ala Lys Tyr Met Leu Glu Glu Ile 610 615 620Ala Lys Leu Thr Gly Cys 625 630 6 609 PRT Xenopus laevis 6 Met Ala LeuPro Gln Leu Ser Glu Gly Ala Ile Ser Ala Met Leu Gly 1 5 10 15 Gly AspSer Ser Cys Lys Pro Thr Leu Gln Val Ile Asn Ile Arg Pro 20 25 30 Ile AsnThr Gly Asn Gly Pro Pro Arg Tyr Arg Leu Leu Met Ser Asp 35 40 45 Gly LeuAsn Thr Leu Ser Ser Phe Met Leu Ala Thr Gln Leu Asn Ser 50 55 60 Leu ValAsp Asn Asn Leu Leu Ala Thr Asn Cys Ile Cys Gln Val Ser 65 70 75 80 ArgPhe Ile Val Asn Asn Leu Lys Asp Gly Arg Arg Val Ile Ile Val 85 90 95 MetGlu Leu Asp Val Leu Lys Ser Ala Asp Leu Val Met Gly Lys Ile 100 105 110Gly Asn Pro Gln Pro Tyr Asn Asp Gly Gln Pro Gln Pro Ala Ala Pro 115 120125 Ala Pro Ala Ser Ala Pro Ala Pro Ala Pro Ser Lys Leu Gln Asn Asn 130135 140 Ser Ala Pro Pro Pro Ser Met Asn Arg Gly Thr Ser Lys Leu Phe Gly145 150 155 160 Gly Gly Ser Leu Leu Asn Thr Pro Gly Gly Ser Gln Ser LysVal Val 165 170 175 Pro Ile Ala Ser Leu Asn Pro Tyr Gln Ser Lys Trp ThrVal Arg Ala 180 185 190 Arg Val Thr Asn Lys Gly Gln Ile Arg Thr Trp SerAsn Ser Arg Gly 195 200 205 Glu Gly Lys Leu Phe Ser Ile Glu Met Val AspGlu Ser Gly Glu Ile 210 215 220 Arg Ala Thr Ala Phe Asn Glu Gln Ala AspLys Phe Phe Ser Ile Ile 225 230 235 240 Glu Val Asn Lys Val Tyr Tyr PheSer Lys Gly Thr Leu Lys Ile Ala 245 250 255 Asn Lys Gln Tyr Thr Ser ValLys Asn Asp Tyr Glu Met Thr Phe Asn 260 265 270 Ser Glu Thr Ser Val IlePro Cys Asp Asp Ser Ala Asp Val Pro Met 275 280 285 Val Gln Phe Glu PheVal Ser Ile Gly Glu Leu Glu Ser Lys Asn Lys 290 295 300 Asp Thr Val LeuAsp Ile Ile Gly Val Cys Lys Asn Val Glu Glu Val 305 310 315 320 Thr LysVal Thr Ile Lys Ser Asn Asn Arg Glu Val Ser Lys Arg Ser 325 330 335 IleHis Leu Met Asp Ser Ser Gly Lys Val Val Ser Thr Thr Leu Trp 340 345 350Gly Glu Asp Ala Asp Lys Phe Asp Gly Ser Arg Gln Pro Val Val Ala 355 360365 Ile Lys Gly Ala Arg Leu Ser Asp Phe Gly Gly Arg Ser Leu Ser Val 370375 380 Leu Ser Ser Ser Thr Val Met Ile Asn Pro Asp Ile Pro Glu Ala Phe385 390 395 400 Lys Leu Arg Ala Trp Phe Asp Ser Glu Gly Gln Val Val GluGly Thr 405 410 415 Ser Ile Ser Glu Ser Arg Gly Gly Gly Thr Gly Gly GlyAsn Thr Asn 420 425 430 Trp Lys Ser Leu Leu Glu Val Lys Asn Glu Asn LeuGly His Gly Glu 435 440 445 Lys Ala Asp Tyr Phe Thr Ser Val Ala Thr IleVal Tyr Leu Arg Lys 450 455 460 Glu Asn Cys Leu Tyr Gln Ala Cys Pro SerGln Asp Cys Asn Lys Lys 465 470 475 480 Val Ile Asp Gln Gln Asn Gly LeuPhe Arg Cys Glu Lys Cys Asn Lys 485 490 495 Glu Phe Pro Asn Phe Lys TyrArg Leu Ile Leu Ser Ala Asn Ile Ala 500 505 510 Asp Phe Gly Glu Asn GlnTrp Ile Thr Cys Phe Gln Glu Ser Ala Glu 515 520 525 Ser Ile Leu Gly GlnAsn Ala Thr Tyr Leu Gly Glu Leu Lys Glu Lys 530 535 540 Asn Glu Gln AlaTyr Asp Glu Val Phe Gln Asn Ala Asn Phe Arg Ser 545 550 555 560 Tyr ThrPhe Arg Ala Arg Val Lys Leu Glu Thr Tyr Asn Asp Glu Ser 565 570 575 ArgIle Lys Ala Thr Ala Val Asp Val Lys Pro Val Asp His Lys Glu 580 585 590Tyr Ser Arg Arg Leu Ile Met Asn Ile Arg Lys Met Ala Thr Gln Gly 595 600605 Val 7 616 PRT Homo sapiens 7 Met Val Gly Gln Leu Ser Glu Gly Ala IleAla Ala Ile Met Gln Lys 1 5 10 15 Gly Asp Thr Asn Ile Lys Pro Ile LeuGln Val Ile Asn Ile Arg Pro 20 25 30 Ile Thr Thr Gly Asn Ser Pro Pro ArgTyr Arg Leu Leu Met Ser Asp 35 40 45 Gly Leu Asn Thr Leu Ser Ser Phe MetLeu Ala Thr Gln Leu Asn Pro 50 55 60 Leu Val Glu Glu Glu Gln Leu Ser SerAsn Cys Val Cys Gln Ile His 65 70 75 80 Arg Phe Ile Val Asn Thr Leu LysAsp Gly Arg Arg Val Val Ile Leu 85 90 95 Met Glu Leu Glu Val Leu Lys SerAla Glu Ala Val Gly Val Lys Ile 100 105 110 Gly Asn Pro Val Pro Tyr AsnGlu Gly Leu Gly Gln Pro Gln Val Ala 115 120 125 Pro Pro Ala Pro Ala AlaSer Pro Ala Ala Ser Ser Arg Pro Gln Pro 130 135 140 Gln Asn Gly Ser SerGly Met Gly Ser Thr Val Ser Lys Ala Tyr Gly 145 150 155 160 Ala Ser LysThr Phe Gly Lys Ala Ala Gly Pro Ser Leu Ser His Thr 165 170 175 Ser GlyGly Thr Gln Ser Lys Val Val Pro Ile Ala Ser Leu Thr Pro 180 185 190 TyrGln Ser Lys Trp Thr Ile Cys Ala Arg Val Thr Asn Lys Ser Gln 195 200 205Ile Arg Thr Trp Ser Asn Ser Arg Gly Glu Gly Lys Leu Phe Ser Leu 210 215220 Glu Leu Val Asp Glu Ser Gly Glu Ile Arg Ala Thr Ala Phe Asn Glu 225230 235 240 Gln Val Asp Lys Phe Phe Pro Leu Ile Glu Val Asn Lys Val TyrTyr 245 250 255 Phe Ser Lys Gly Thr Leu Lys Ile Ala Asn Lys Gln Phe ThrAla Val 260 265 270 Lys Asn Asp Tyr Glu Met Thr Phe Asn Asn Glu Thr SerVal Met Pro 275 280 285 Cys Glu Asp Asp His His Leu Pro Thr Val Gln PheAsp Phe Thr Gly 290 295 300 Ile Asp Asp Leu Glu Asn Lys Ser Lys Asp SerLeu Val Asp Ile Ile 305 310 315 320 Gly Ile Cys Lys Ser Tyr Glu Asp AlaThr Lys Ile Thr Val Arg Ser 325 330 335 Asn Asn Arg Glu Val Ala Lys ArgAsn Ile Tyr Leu Met Asp Thr Ser 340 345 350 Gly Lys Val Val Thr Ala ThrLeu Trp Gly Glu Asp Ala Asp Lys Phe 355 360 365 Asp Gly Ser Arg Gln ProVal Leu Ala Ile Lys Gly Ala Arg Val Ser 370 375 380 Asp Phe Gly Gly ArgSer Leu Ser Val Leu Ser Ser Ser Thr Ile Ile 385 390 395 400 Ala Asn ProAsp Ile Pro Glu Ala Tyr Lys Leu Arg Gly Trp Phe Asp 405 410 415 Ala GluGly Gln Ala Leu Asp Gly Val Ser Ile Ser Asp Leu Lys Ser 420 425 430 GlyGly Val Gly Gly Ser Asn Thr Asn Trp Lys Thr Leu Tyr Glu Val 435 440 445Lys Ser Glu Asn Leu Gly Gln Gly Asp Lys Pro Asp Tyr Phe Ser Ser 450 455460 Val Ala Thr Val Val Tyr Leu Arg Lys Glu Asn Cys Met Tyr Gln Ala 465470 475 480 Cys Pro Thr Gln Asp Cys Asn Lys Lys Val Ile Asp Gln Gln AsnGly 485 490 495 Leu Tyr Arg Cys Glu Lys Cys Asp Thr Glu Phe Pro Asn PheLys Tyr 500 505 510 Arg Met Ile Leu Ser Val Asn Ile Ala Asp Phe Gln GluAsn Gln Trp 515 520 525 Val Thr Cys Phe Gln Glu Ser Ala Glu Ala Ile LeuGly Gln Asn Ala 530 535 540 Ala Tyr Leu Gly Glu Leu Lys Asp Lys Asn GluGln Ala Phe Glu Glu 545 550 555 560 Val Phe Gln Asn Ala Asn Phe Arg SerPhe Ile Phe Arg Val Arg Val 565 570 575 Lys Val Glu Thr Tyr Asn Asp GluSer Arg Ile Lys Ala Thr Val Met 580 585 590 Asp Val Lys Pro Val Asp TyrArg Glu Tyr Gly Arg Arg Leu Val Met 595 600 605 Ser Ile Arg Arg Ser AlaLeu Met 610 615 8 603 PRT Drosophila melanogaster 8 Met Val Leu Ala SerLeu Ser Thr Gly Val Ile Ala Arg Ile Met His 1 5 10 15 Gly Glu Val ValAsp Ala Pro Val Leu Gln Ile Leu Ala Ile Lys Lys 20 25 30 Ile Asn Ser AlaAla Asp Ser Glu Arg Tyr Arg Ile Leu Ile Ser Asp 35 40 45 Gly Lys Tyr PheAsn Ser Tyr Ala Met Leu Ala Ser Gln Leu Asn Val 50 55 60 Met Gln His AsnGly Glu Leu Glu Glu Phe Thr Ile Val Gln Leu Asp 65 70 75 80 Lys Tyr ValThr Ser Leu Val Gly Lys Asp Gly Ala Gly Lys Arg Val 85 90 95 Leu Ile IleSer Glu Leu Thr Val Val Asn Pro Gly Ala Glu Val Lys 100 105 110 Ser LysIle Gly Glu Pro Val Thr Tyr Glu Asn Ala Ala Lys Gln Asp 115 120 125 LeuAla Pro Lys Pro Ala Val Thr Ser Asn Ser Lys Pro Ile Ala Lys 130 135 140Lys Glu Pro Ser His Asn Asn Asn Asn Asn Ile Val Met Asn Ser Ser 145 150155 160 Ile Asn Ser Gly Met Thr His Pro Ile Ser Ser Leu Ser Pro Tyr Gln165 170 175 Asn Lys Trp Val Ile Lys Ala Arg Val Thr Ser Lys Ser Gly IleArg 180 185 190 Thr Trp Ser Asn Ala Arg Gly Glu Gly Lys Leu Phe Ser MetAsp Leu 195 200 205 Met Asp Glu Ser Gly Glu Ile Arg Ala Thr Ala Phe LysGlu Gln Cys 210 215 220 Asp Lys Phe Tyr Asp Leu Ile Gln Val Asp Ser ValTyr Tyr Ile Ser 225 230 235 240 Lys Cys Gln Leu Lys Pro Ala Asn Lys GlnTyr Ser Ser Leu Asn Asn 245 250 255 Ala Tyr Glu Met Thr Phe Ser Gly GluThr Val Val Gln Leu Cys Glu 260 265 270 Asp Thr Asp Asp Asp Pro Ile ProGlu Ile Lys Tyr Asn Leu Val Pro 275 280 285 Ile Ser Asp Val Ser Gly MetGlu Asn Lys Ala Ala Val Asp Thr Ile 290 295 300 Gly Ile Cys Lys Glu ValGly Glu Leu Gln Ser Phe Val Ala Arg Thr 305 310 315 320 Thr Asn Lys GluPhe Lys Lys Arg Asp Ile Thr Leu Val Asp Met Ser 325 330 335 Asn Ser AlaIle Ser Leu Thr Leu Trp Gly Asp Asp Ala Val Asn Phe 340 345 350 Asp GlyHis Val Gln Pro Val Ile Leu Val Lys Gly Thr Arg Ile Asn 355 360 365 GluPhe Asn Gly Gly Lys Ser Leu Ser Leu Gly Gly Gly Ser Ile Met 370 375 380Lys Ile Asn Pro Asp Ile Pro Glu Ala His Lys Leu Arg Gly Trp Phe 385 390395 400 Asp Asn Gly Gly Gly Asp Ser Val Ala Asn Met Val Ser Ala Arg Thr405 410 415 Gly Gly Gly Ser Phe Ser Thr Glu Trp Met Thr Leu Lys Asp AlaArg 420 425 430 Ala Arg Asn Leu Gly Ser Gly Asp Lys Pro Asp Tyr Phe GlnCys Lys 435 440 445 Ala Val Val His Ile Val Lys Gln Glu Asn Ala Phe TyrArg Ala Cys 450 455 460 Pro Gln Ser Asp Cys Asn Lys Lys Val Val Asp GluGly Asn Asp Gln 465 470 475 480 Phe Arg Cys Glu Lys Cys Asn Ala Leu PhePro Asn Phe Lys Tyr Arg 485 490 495 Leu Leu Ile Asn Met Ser Ile Gly AspTrp Thr Ser Asn Arg Trp Val 500 505 510 Ser Ser Phe Asn Glu Val Gly GluGln Leu Leu Gly His Thr Ser Gln 515 520 525 Glu Val Gly Glu Ala Leu GluAsn Asp Pro Ala Lys Ala Glu Gln Ile 530 535 540 Phe Ser Ala Leu Asn PheThr Ser His Ile Phe Lys Leu Arg Cys Lys 545 550 555 560 Asn Glu Val TyrGly Asp Met Thr Arg Asn Lys Leu Thr Val Gln Ser 565 570 575 Val Ala ProIle Asn His Lys Glu Tyr Asn Lys His Leu Leu Lys Glu 580 585 590 Leu GlnGlu Leu Thr Gly Ile Gly Ser Ser Asn 595 600 9 609 PRTSchizosaccharomyces pombe 9 Met Ala Glu Arg Leu Ser Val Gly Ala Leu ArgIle Ile Asn Thr Ser 1 5 10 15 Asp Ala Ser Ser Phe Pro Pro Asn Pro IleLeu Gln Val Leu Thr Val 20 25 30 Lys Glu Leu Asn Ser Asn Pro Thr Ser GlyAla Pro Lys Arg Tyr Arg 35 40 45 Val Val Leu Ser Asp Ser Ile Asn Tyr AlaGln Ser Met Leu Ser Thr 50 55 60 Gln Leu Asn His Leu Val Ala Glu Asn LysLeu Gln Lys Gly Ala Phe 65 70 75 80 Val Gln Leu Thr Gln Phe Thr Val AsnVal Met Lys Glu Arg Lys Ile 85 90 95 Leu Ile Val Leu Gly Leu Asn Val LeuThr Glu Leu Gly Val Met Asp 100 105 110 Lys Ile Gly Asn Pro Ala Gly LeuGlu Thr Val Asp Ala Leu Arg Gln 115 120 125 Gln Gln Asn Glu Gln Asn AsnAla Ser Ala Pro Arg Thr Gly Ile Ser 130 135 140 Thr Ser Thr Asn Ser PheTyr Gly Asn Asn Ala Ala Ala Thr Ala Pro 145 150 155 160 Ala Pro Pro ProMet Met Lys Lys Pro Ala Ala Pro Asn Ser Leu Ser 165 170 175 Thr Ile IleTyr Pro Ile Glu Gly Leu Ser Pro Tyr Gln Asn Lys Trp 180 185 190 Thr IleArg Ala Arg Val Thr Asn Lys Ser Glu Val Lys His Trp His 195 200 205 AsnGln Arg Gly Glu Gly Lys Leu Phe Ser Val Asn Leu Leu Asp Glu 210 215 220Ser Gly Glu Ile Arg Ala Thr Gly Phe Asn Asp Gln Val Asp Ala Phe 225 230235 240 Tyr Asp Ile Leu Gln Glu Gly Ser Val Tyr Tyr Ile Ser Arg Cys Arg245 250 255 Val Asn Ile Ala Lys Lys Gln Tyr Thr Asn Val Gln Asn Glu TyrGlu 260 265 270 Leu Met Phe Glu Arg Asp Thr Glu Ile Arg Lys Ala Glu AspGln Thr 275 280 285 Ala Val Pro Val Ala Lys Phe Ser Phe Val Ser Leu GlnGlu Val Gly 290 295 300 Asp Val Ala Lys Asp Ala Val Ile Asp Val Ile GlyVal Leu Gln Asn 305 310 315 320 Val Gly Pro Val Gln Gln Ile Thr Ser ArgAla Thr Ser Arg Gly Phe 325 330 335 Asp Lys Arg Asp Ile Thr Ile Val AspGln Thr Gly Tyr Glu Met Arg 340 345 350 Val Thr Leu Trp Gly Lys Thr AlaIle Glu Phe Ser Val Ser Glu Glu 355 360 365 Ser Ile Leu Ala Phe Lys GlyVal Lys Val Asn Asp Phe Gln Gly Arg 370 375 380 Ser Leu Ser Met Leu ThrSer Ser Thr Met Ser Val Asp Pro Asp Ile 385 390 395 400 Gln Glu Ser HisLeu Leu Lys Gly Trp Tyr Asp Gly Gln Gly Arg Gly 405 410 415 Gln Glu PheAla Lys His Ser Val Ile Ser Ser Thr Leu Ser Thr Thr 420 425 430 Gly ArgSer Ala Glu Arg Lys Asn Ile Ala Glu Val Gln Ala Glu His 435 440 445 LeuGly Met Ser Glu Thr Pro Asp Tyr Phe Ser Leu Lys Gly Thr Ile 450 455 460Val Tyr Ile Arg Lys Lys Asn Val Ser Tyr Pro Ala Cys Pro Ala Ala 465 470475 480 Asp Cys Asn Lys Lys Val Phe Asp Gln Gly Gly Ser Trp Arg Cys Glu485 490 495 Lys Cys Asn Lys Glu Tyr Asp Ala Pro Gln Tyr Arg Tyr Ile IleThr 500 505 510 Ile Ala Val Gly Asp His Thr Gly Gln Leu Trp Leu Asn ValPhe Asp 515 520 525 Asp Val Gly Lys Leu Ile Met His Lys Thr Ala Asp GluLeu Asn Asp 530 535 540 Leu Gln Glu Asn Asp Glu Asn Ala Phe Met Asn CysMet Ala Glu Ala 545 550 555 560 Cys Tyr Met Pro Tyr Ile Phe Gln Cys ArgAla Lys Gln Asp Asn Phe 565 570 575 Lys Gly Glu Met Arg Val Arg Tyr ThrVal Met Ser Ile Asn Gln Met 580 585 590 Asp Trp Lys Glu Glu Ser Lys ArgLeu Ile Asn Phe Ile Glu Ser Ala 595 600 605 Gln 10 621 PRT Saccharomycescerevisiae 10 Met Ser Ser Val Gln Leu Ser Arg Gly Asp Phe His Ser IlePhe Thr 1 5 10 15 Asn Lys Gln Arg Tyr Asp Asn Pro Thr Gly Gly Val TyrGln Val Tyr 20 25 30 Asn Thr Arg Lys Ser Asp Gly Ala Asn Ser Asn Arg LysAsn Leu Ile 35 40 45 Met Ile Ser Asp Gly Ile Tyr His Met Lys Ala Leu LeuArg Asn Gln 50 55 60 Ala Ala Ser Lys Phe Gln Ser Met Glu Leu Gln Arg GlyAsp Ile Ile 65 70 75 80 Arg Val Ile Ile Ala Glu Pro Ala Ile Val Arg GluArg Lys Lys Tyr 85 90 95 Val Leu Leu Val Asp Asp Phe Glu Leu Val Gln SerArg Ala Asp Met 100 105 110 Val Asn Gln Thr Ser Thr Phe Leu Asp Asn TyrPhe Ser Glu His Pro 115 120 125 Asn Glu Thr Leu Lys Asp Glu Asp Ile ThrAsp Ser Gly Asn Val Ala 130 135 140 Asn Gln Thr Asn Ala Ser Asn Ala GlyVal Pro Asp Met Leu His Ser 145 150 155 160 Asn Ser Asn Leu Asn Ala AsnGlu Arg Lys Phe Ala Asn Glu Asn Pro 165 170 175 Asn Ser Gln Lys Thr ArgPro Ile Phe Ala Ile Glu Gln Leu Ser Pro 180 185 190 Tyr Gln Asn Val TrpThr Ile Lys Ala Arg Val Ser Tyr Lys Gly Glu 195 200 205 Ile Lys Thr TrpHis Asn Gln Arg Gly Asp Gly Lys Leu Phe Asn Val 210 215 220 Asn Phe LeuAsp Thr Ser Gly Glu Ile Arg Ala Thr Ala Phe Asn Asp 225 230 235 240 PheAla Thr Lys Phe Asn Glu Ile Leu Gln Glu Gly Lys Val Tyr Tyr 245 250 255Val Ser Lys Ala Lys Leu Gln Pro Ala Lys Pro Gln Phe Thr Asn Leu 260 265270 Thr His Pro Tyr Glu Leu Asn Leu Asp Arg Asp Thr Val Ile Glu Glu 275280 285 Cys Phe Asp Glu Ser Asn Val Pro Lys Thr His Phe Asn Phe Ile Lys290 295 300 Leu Asp Ala Ile Gln Asn Gln Glu Val Asn Ser Asn Val Asp ValLeu 305 310 315 320 Gly Ile Ile Gln Thr Ile Asn Pro His Phe Glu Leu ThrSer Arg Ala 325 330 335 Gly Lys Lys Phe Asp Arg Arg Asp Ile Thr Ile ValAsp Asp Ser Gly 340 345 350 Phe Ser Ile Ser Val Gly Leu Trp Asn Gln GlnAla Leu Asp Phe Asn 355 360 365 Leu Pro Glu Gly Ser Val Ala Ala Ile LysGly Val Arg Val Thr Asp 370 375 380 Phe Gly Gly Lys Ser Leu Ser Met GlyPhe Ser Ser Thr Leu Ile Pro 385 390 395 400 Asn Pro Glu Ile Pro Glu AlaTyr Ala Leu Lys Gly Trp Tyr Asp Ser 405 410 415 Lys Gly Arg Asn Ala AsnPhe Ile Thr Leu Lys Gln Glu Pro Gly Met 420 425 430 Gly Gly Gln Ser AlaAla Ser Leu Thr Lys Phe Ile Ala Gln Arg Ile 435 440 445 Thr Ile Ala ArgAla Gln Ala Glu Asn Leu Gly Arg Ser Glu Lys Gly 450 455 460 Asp Phe PheSer Val Lys Ala Ala Ile Ser Phe Leu Lys Val Asp Asn 465 470 475 480 PheAla Tyr Pro Ala Cys Ser Asn Glu Asn Cys Asn Lys Lys Val Leu 485 490 495Glu Gln Pro Asp Gly Thr Trp Arg Cys Glu Lys Cys Asp Thr Asn Asn 500 505510 Ala Arg Pro Asn Trp Arg Tyr Ile Leu Thr Ile Ser Ile Ile Asp Glu 515520 525 Thr Asn Gln Leu Trp Leu Thr Leu Phe Asp Asp Gln Ala Lys Gln Leu530 535 540 Leu Gly Val Asp Ala Asn Thr Leu Met Ser Leu Lys Glu Glu AspPro 545 550 555 560 Asn Glu Phe Thr Lys Ile Thr Gln Ser Ile Gln Met AsnGlu Tyr Asp 565 570 575 Phe Arg Ile Arg Ala Arg Glu Asp Thr Tyr Asn AspGln Ser Arg Ile 580 585 590 Arg Tyr Thr Val Ala Asn Leu His Ser Leu AsnTyr Arg Ala Glu Ala 595 600 605 Asp Tyr Leu Ala Asp Glu Leu Ser Lys AlaLeu Leu Ala 610 615 620 11 1124 DNA Zea mays misc_feature (0)...(0)Maize RPA Middle Subunit Homologue-1 11 tcgacccacg cgtccgatcc tcccatctgcgcacccgcaa gcctattcgc cgcacctcct 60 caggtgaccg ggaag atg atg ccg ttg agccaa acc gac ttc tcg ccg tcg 111 Met Met Pro Leu Ser Gln Thr Asp Phe SerPro Ser 1 5 10 cag ttc acc tcc tcc cag aat gcc gcc gcc gac tcc acc acgcct tcc 159 Gln Phe Thr Ser Ser Gln Asn Ala Ala Ala Asp Ser Thr Thr ProSer 15 20 25 aag atg cgc ggc gcg tcc agc acc atg ccg ctc acc gtg aag caggtc 207 Lys Met Arg Gly Ala Ser Ser Thr Met Pro Leu Thr Val Lys Gln Val30 35 40 gtc gac gcg cag cag tct ggc acg ggc gag aag ggc gct ccg ttc atc255 Val Asp Ala Gln Gln Ser Gly Thr Gly Glu Lys Gly Ala Pro Phe Ile 4550 55 60 gtc aat ggc gtc gag atg gct aac att cga ctt gtg ggg atg gtc aat303 Val Asn Gly Val Glu Met Ala Asn Ile Arg Leu Val Gly Met Val Asn 6570 75 gcc aag gtg gag cgg acg acc gat gtg acc ttc acg ctc gac gat ggc351 Ala Lys Val Glu Arg Thr Thr Asp Val Thr Phe Thr Leu Asp Asp Gly 8085 90 acc ggc cgc ctc gat ttc atc aga tgg gtg aat gat gct tca gat tct399 Thr Gly Arg Leu Asp Phe Ile Arg Trp Val Asn Asp Ala Ser Asp Ser 95100 105 ttt gaa act gct gct att cag aat ggt atg tac att gcg gtc att gga447 Phe Glu Thr Ala Ala Ile Gln Asn Gly Met Tyr Ile Ala Val Ile Gly 110115 120 agc ctc aag gga ctg caa gag agg aag cgt gct act gct ttc tca atc495 Ser Leu Lys Gly Leu Gln Glu Arg Lys Arg Ala Thr Ala Phe Ser Ile 125130 135 140 agg cct ata acc gat ttc aat gag gtt acg ctg cat ttc att cagtgt 543 Arg Pro Ile Thr Asp Phe Asn Glu Val Thr Leu His Phe Ile Gln Cys145 150 155 gtt cgg atg cat ata gag aac att gaa tta aag gct ggc agt cctgca 591 Val Arg Met His Ile Glu Asn Ile Glu Leu Lys Ala Gly Ser Pro Ala160 165 170 cga atc agt tct tct atg gga gtg tca ttc tca aat gga ttc agtgaa 639 Arg Ile Ser Ser Ser Met Gly Val Ser Phe Ser Asn Gly Phe Ser Glu175 180 185 tca agc aca ccg aca tct ttg aaa tcc agt ccc gca ccg gtg accagc 687 Ser Ser Thr Pro Thr Ser Leu Lys Ser Ser Pro Ala Pro Val Thr Ser190 195 200 ggg tca tcc gat act gat ctg cac acg cag gtc ctg aat ttt tttaat 735 Gly Ser Ser Asp Thr Asp Leu His Thr Gln Val Leu Asn Phe Phe Asn205 210 215 220 gaa cca gcg aac ctc gag agt gag cat ggg gtg cac gtt gatgaa gta 783 Glu Pro Ala Asn Leu Glu Ser Glu His Gly Val His Val Asp GluVal 225 230 235 ctc aag cgg ttc aaa ctt ttg ccg aag aag cag atc acg gatgct att 831 Leu Lys Arg Phe Lys Leu Leu Pro Lys Lys Gln Ile Thr Asp AlaIle 240 245 250 gat tac aat atg gac tcg ggg cgt ctt tac tca aca att gatgaa ttc 879 Asp Tyr Asn Met Asp Ser Gly Arg Leu Tyr Ser Thr Ile Asp GluPhe 255 260 265 cac tac aag gca act taaccgattt gaaggccagc ctgctggaaatggcagagga 934 His Tyr Lys Ala Thr 270 ctaagtatca cttgtactaa accaaagtctggaaatgtca tgttgtgtca tgaaatgcat 994 ggttggttta tggaaacatt tatatcttgtatcaactagt tgatttgtat ctcgtgtcaa 1054 cttaatgact gagccaagaa aaggaagatgtagaggccga cagaaaaaaa aaaaaaaaaa 1114 aaaaaaaaaa 1124 12 273 PRT Zeamays 12 Met Met Pro Leu Ser Gln Thr Asp Phe Ser Pro Ser Gln Phe Thr Ser1 5 10 15 Ser Gln Asn Ala Ala Ala Asp Ser Thr Thr Pro Ser Lys Met ArgGly 20 25 30 Ala Ser Ser Thr Met Pro Leu Thr Val Lys Gln Val Val Asp AlaGln 35 40 45 Gln Ser Gly Thr Gly Glu Lys Gly Ala Pro Phe Ile Val Asn GlyVal 50 55 60 Glu Met Ala Asn Ile Arg Leu Val Gly Met Val Asn Ala Lys ValGlu 65 70 75 80 Arg Thr Thr Asp Val Thr Phe Thr Leu Asp Asp Gly Thr GlyArg Leu 85 90 95 Asp Phe Ile Arg Trp Val Asn Asp Ala Ser Asp Ser Phe GluThr Ala 100 105 110 Ala Ile Gln Asn Gly Met Tyr Ile Ala Val Ile Gly SerLeu Lys Gly 115 120 125 Leu Gln Glu Arg Lys Arg Ala Thr Ala Phe Ser IleArg Pro Ile Thr 130 135 140 Asp Phe Asn Glu Val Thr Leu His Phe Ile GlnCys Val Arg Met His 145 150 155 160 Ile Glu Asn Ile Glu Leu Lys Ala GlySer Pro Ala Arg Ile Ser Ser 165 170 175 Ser Met Gly Val Ser Phe Ser AsnGly Phe Ser Glu Ser Ser Thr Pro 180 185 190 Thr Ser Leu Lys Ser Ser ProAla Pro Val Thr Ser Gly Ser Ser Asp 195 200 205 Thr Asp Leu His Thr GlnVal Leu Asn Phe Phe Asn Glu Pro Ala Asn 210 215 220 Leu Glu Ser Glu HisGly Val His Val Asp Glu Val Leu Lys Arg Phe 225 230 235 240 Lys Leu LeuPro Lys Lys Gln Ile Thr Asp Ala Ile Asp Tyr Asn Met 245 250 255 Asp SerGly Arg Leu Tyr Ser Thr Ile Asp Glu Phe His Tyr Lys Ala 260 265 270 Thr13 979 DNA Zea mays misc_feature (0)...(0) Maize RPA Middle SubunitHomologue-2 and 3 13 ttcggcacga gcgcacctcc tcaggtgacc gggaag atg atg ccgttg agc caa 54 Met Met Pro Leu Ser Gln 1 5 acc gac ttc tcg ccg tcg cagttc acc tcc tcc cag aat gcc gcc gcc 102 Thr Asp Phe Ser Pro Ser Gln PheThr Ser Ser Gln Asn Ala Ala Ala 10 15 20 gac tcc acc acg cct tcc aag atgcgc ggc gcg tcc agc acc atg ccg 150 Asp Ser Thr Thr Pro Ser Lys Met ArgGly Ala Ser Ser Thr Met Pro 25 30 35 ctc acc gtg aag cag gtc gtc gac gcgcag cag tct ggc acg ggc gac 198 Leu Thr Val Lys Gln Val Val Asp Ala GlnGln Ser Gly Thr Gly Asp 40 45 50 aag ggc gct ccg ttc atc gtc aat ggc gtcgag atg gct aac att cga 246 Lys Gly Ala Pro Phe Ile Val Asn Gly Val GluMet Ala Asn Ile Arg 55 60 65 70 ctt gtg ggg atg gtc aat gcc aag gtg gagcgg acg acc gat gtg acc 294 Leu Val Gly Met Val Asn Ala Lys Val Glu ArgThr Thr Asp Val Thr 75 80 85 ttc acg ctc gac gat ggc acc ggc cgc ctc gatttc atc aga tgg gtg 342 Phe Thr Leu Asp Asp Gly Thr Gly Arg Leu Asp PheIle Arg Trp Val 90 95 100 aat gat gct tca gat tct ttt gaa act gct gctatt cag aat ggt atg 390 Asn Asp Ala Ser Asp Ser Phe Glu Thr Ala Ala IleGln Asn Gly Met 105 110 115 tac att gcg gtc att gga agc ctc aag gga ctgcaa gag agg aag cgt 438 Tyr Ile Ala Val Ile Gly Ser Leu Lys Gly Leu GlnGlu Arg Lys Arg 120 125 130 gct act gct ttc tca atc agg cct ata acc gatttc aat gag gtt acg 486 Ala Thr Ala Phe Ser Ile Arg Pro Ile Thr Asp PheAsn Glu Val Thr 135 140 145 150 ctg cat ttc att cag tgt gtt cgg atg catata gag aac att gaa tta 534 Leu His Phe Ile Gln Cys Val Arg Met His IleGlu Asn Ile Glu Leu 155 160 165 aag gct ggc agt cct gca cga atc agt tcttct atg gga gtg tca ttc 582 Lys Ala Gly Ser Pro Ala Arg Ile Ser Ser SerMet Gly Val Ser Phe 170 175 180 tca aat gga ttc agt gaa tca agc aca ccgaca tct ttg aaa tcc agt 630 Ser Asn Gly Phe Ser Glu Ser Ser Thr Pro ThrSer Leu Lys Ser Ser 185 190 195 ccc gca ccg gtg acc agc ggg tca tcc gatact gat ctg cac acg cag 678 Pro Ala Pro Val Thr Ser Gly Ser Ser Asp ThrAsp Leu His Thr Gln 200 205 210 gtc ctg aat ttt ttt aat gaa cca gcg aacctc gag agt gag cat ggg 726 Val Leu Asn Phe Phe Asn Glu Pro Ala Asn LeuGlu Ser Glu His Gly 215 220 225 230 gtg cac gtt gat gaa gta ctc aag cggttc aaa ctt ttg ccg aag aag 774 Val His Val Asp Glu Val Leu Lys Arg PheLys Leu Leu Pro Lys Lys 235 240 245 cag atc acg gat gct att gat tac aatatg gac tcg ggg cgt ctt tac 822 Gln Ile Thr Asp Ala Ile Asp Tyr Asn MetAsp Ser Gly Arg Leu Tyr 250 255 260 tca aca att gat gaa ttc cac tac aaggca act taaccgattt gaaggccagc 875 Ser Thr Ile Asp Glu Phe His Tyr LysAla Thr 265 270 ctgctggaaa tggcagagga ctaagtatca cttgtactaa accaaagtctggaaatgtca 935 tgttgtgtca tgaaatgcat ggttggttta tggaaacaaa aaaa 979 14273 PRT Zea mays 14 Met Met Pro Leu Ser Gln Thr Asp Phe Ser Pro Ser GlnPhe Thr Ser 1 5 10 15 Ser Gln Asn Ala Ala Ala Asp Ser Thr Thr Pro SerLys Met Arg Gly 20 25 30 Ala Ser Ser Thr Met Pro Leu Thr Val Lys Gln ValVal Asp Ala Gln 35 40 45 Gln Ser Gly Thr Gly Asp Lys Gly Ala Pro Phe IleVal Asn Gly Val 50 55 60 Glu Met Ala Asn Ile Arg Leu Val Gly Met Val AsnAla Lys Val Glu 65 70 75 80 Arg Thr Thr Asp Val Thr Phe Thr Leu Asp AspGly Thr Gly Arg Leu 85 90 95 Asp Phe Ile Arg Trp Val Asn Asp Ala Ser AspSer Phe Glu Thr Ala 100 105 110 Ala Ile Gln Asn Gly Met Tyr Ile Ala ValIle Gly Ser Leu Lys Gly 115 120 125 Leu Gln Glu Arg Lys Arg Ala Thr AlaPhe Ser Ile Arg Pro Ile Thr 130 135 140 Asp Phe Asn Glu Val Thr Leu HisPhe Ile Gln Cys Val Arg Met His 145 150 155 160 Ile Glu Asn Ile Glu LeuLys Ala Gly Ser Pro Ala Arg Ile Ser Ser 165 170 175 Ser Met Gly Val SerPhe Ser Asn Gly Phe Ser Glu Ser Ser Thr Pro 180 185 190 Thr Ser Leu LysSer Ser Pro Ala Pro Val Thr Ser Gly Ser Ser Asp 195 200 205 Thr Asp LeuHis Thr Gln Val Leu Asn Phe Phe Asn Glu Pro Ala Asn 210 215 220 Leu GluSer Glu His Gly Val His Val Asp Glu Val Leu Lys Arg Phe 225 230 235 240Lys Leu Leu Pro Lys Lys Gln Ile Thr Asp Ala Ile Asp Tyr Asn Met 245 250255 Asp Ser Gly Arg Leu Tyr Ser Thr Ile Asp Glu Phe His Tyr Lys Ala 260265 270 Thr 15 1051 DNA Zea mays misc_feature (0)...(0) Maize RPA MiddleSubunit Homologue-4 15 tcgacccacg cgtccgatcc tcccatctgc gcacccgcaagcctattcgc cgcacctcct 60 caggtgaccg ggaag atg atg ccg ttg agc caa accgac ttc tcg ccg tcg 111 Met Met Pro Leu Ser Gln Thr Asp Phe Ser Pro Ser1 5 10 cag ttc acc tcc tcc cag aat gcc gcc gcc gac tcc acc acg cct tcc159 Gln Phe Thr Ser Ser Gln Asn Ala Ala Ala Asp Ser Thr Thr Pro Ser 1520 25 aag atg cgc ggc gcg tcc agc acc atg ccg ctc acc gtg aag cag gtc207 Lys Met Arg Gly Ala Ser Ser Thr Met Pro Leu Thr Val Lys Gln Val 3035 40 gtc gac gcg cag cag tct ggc acg ggc gag aag ggc gct ccg ttc atc255 Val Asp Ala Gln Gln Ser Gly Thr Gly Glu Lys Gly Ala Pro Phe Ile 4550 55 60 gtc aat ggc gtc gag atg gct aac att cga ctt gtg ggg atg gtc aat303 Val Asn Gly Val Glu Met Ala Asn Ile Arg Leu Val Gly Met Val Asn 6570 75 gcc aag gtg gag cgg acg acc gat gtg acc ttc acg ctc gac gat ggc351 Ala Lys Val Glu Arg Thr Thr Asp Val Thr Phe Thr Leu Asp Asp Gly 8085 90 acc ggc cgc ctc gat ttc atc aga tgg gtg aat gat gct tca gat tct399 Thr Gly Arg Leu Asp Phe Ile Arg Trp Val Asn Asp Ala Ser Asp Ser 95100 105 ttt gaa act gct gct att cag aat ggt atg tac att gcg gtc att gga447 Phe Glu Thr Ala Ala Ile Gln Asn Gly Met Tyr Ile Ala Val Ile Gly 110115 120 agc ctc aag gga ctg caa gag agg aag cgt gct act gct ttc tca atc495 Ser Leu Lys Gly Leu Gln Glu Arg Lys Arg Ala Thr Ala Phe Ser Ile 125130 135 140 agg cct ata acc gat ttc aat gag gtt acg ctg cat ttc att cagtgt 543 Arg Pro Ile Thr Asp Phe Asn Glu Val Thr Leu His Phe Ile Gln Cys145 150 155 gtt cgg atg cat ata gag aac act gaa tta aag gct ggc agt cctgca 591 Val Arg Met His Ile Glu Asn Thr Glu Leu Lys Ala Gly Ser Pro Ala160 165 170 cga atc aat tct tct atg gga gtg tca ttc tca aat gga ttc agtgaa 639 Arg Ile Asn Ser Ser Met Gly Val Ser Phe Ser Asn Gly Phe Ser Glu175 180 185 tca agc aca ccg aca tct ttg aaa tcc agt ccc gca ccg gtg accagc 687 Ser Ser Thr Pro Thr Ser Leu Lys Ser Ser Pro Ala Pro Val Thr Ser190 195 200 ggg tca tcc gat act gat ctg cac acg cag gtc ctg aat ttt tttaat 735 Gly Ser Ser Asp Thr Asp Leu His Thr Gln Val Leu Asn Phe Phe Asn205 210 215 220 gaa cca gcg aac ctc gag agt gag cat ggg gtg cac gtt gatgaa gta 783 Glu Pro Ala Asn Leu Glu Ser Glu His Gly Val His Val Asp GluVal 225 230 235 ctc aag cgg ttc aaa ctt ttg ccg aag aag cag atc acg gatgct att 831 Leu Lys Arg Phe Lys Leu Leu Pro Lys Lys Gln Ile Thr Asp AlaIle 240 245 250 gat tac aat atg gac tcg ggg cgt ctt tac tca aca att gatgaa ttc 879 Asp Tyr Asn Met Asp Ser Gly Arg Leu Tyr Ser Thr Ile Asp GluPhe 255 260 265 cac tac aag gca act taaccgattt gaaggtcagc ctgctggaaatggcagagga 934 His Tyr Lys Ala Thr 270 ctaagtatca cttgtactaa accaaagtctggaaatgtca tgttgtgtca tgaaatgcat 994 ggttggttta tggaaacaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaa 1051 16 273 PRT Zea mays 16 Met Met ProLeu Ser Gln Thr Asp Phe Ser Pro Ser Gln Phe Thr Ser 1 5 10 15 Ser GlnAsn Ala Ala Ala Asp Ser Thr Thr Pro Ser Lys Met Arg Gly 20 25 30 Ala SerSer Thr Met Pro Leu Thr Val Lys Gln Val Val Asp Ala Gln 35 40 45 Gln SerGly Thr Gly Glu Lys Gly Ala Pro Phe Ile Val Asn Gly Val 50 55 60 Glu MetAla Asn Ile Arg Leu Val Gly Met Val Asn Ala Lys Val Glu 65 70 75 80 ArgThr Thr Asp Val Thr Phe Thr Leu Asp Asp Gly Thr Gly Arg Leu 85 90 95 AspPhe Ile Arg Trp Val Asn Asp Ala Ser Asp Ser Phe Glu Thr Ala 100 105 110Ala Ile Gln Asn Gly Met Tyr Ile Ala Val Ile Gly Ser Leu Lys Gly 115 120125 Leu Gln Glu Arg Lys Arg Ala Thr Ala Phe Ser Ile Arg Pro Ile Thr 130135 140 Asp Phe Asn Glu Val Thr Leu His Phe Ile Gln Cys Val Arg Met His145 150 155 160 Ile Glu Asn Thr Glu Leu Lys Ala Gly Ser Pro Ala Arg IleAsn Ser 165 170 175 Ser Met Gly Val Ser Phe Ser Asn Gly Phe Ser Glu SerSer Thr Pro 180 185 190 Thr Ser Leu Lys Ser Ser Pro Ala Pro Val Thr SerGly Ser Ser Asp 195 200 205 Thr Asp Leu His Thr Gln Val Leu Asn Phe PheAsn Glu Pro Ala Asn 210 215 220 Leu Glu Ser Glu His Gly Val His Val AspGlu Val Leu Lys Arg Phe 225 230 235 240 Lys Leu Leu Pro Lys Lys Gln IleThr Asp Ala Ile Asp Tyr Asn Met 245 250 255 Asp Ser Gly Arg Leu Tyr SerThr Ile Asp Glu Phe His Tyr Lys Ala 260 265 270 Thr 17 1087 DNA Zea maysmisc_feature (0)...(0) Maize RPA Middle Subunit Homologue-5 17aattccgggg ccgacccacg cgtccgcatc gatcctccca tctgcgcacc cgcaagccta 60ttcgccgcac ctcctcaggt gaccgggaag atg atg ccg ttg agc caa acc gac 114 MetMet Pro Leu Ser Gln Thr Asp 1 5 ttc tcg ccg tcg cag ttc acc tcc tcc cagaat gcc gcc gcc gac tcc 162 Phe Ser Pro Ser Gln Phe Thr Ser Ser Gln AsnAla Ala Ala Asp Ser 10 15 20 acc acg cct tcc aag atg cgc ggc gcg tcc agcacc atg ccg ctc acc 210 Thr Thr Pro Ser Lys Met Arg Gly Ala Ser Ser ThrMet Pro Leu Thr 25 30 35 40 gtg aag car gtc gtc gac gcg cag cag tct ggcacg ggc gag aag ggc 258 Val Lys Xaa Val Val Asp Ala Gln Gln Ser Gly ThrGly Glu Lys Gly 45 50 55 gct ccg ttc atc gtc aat ggc gtc gag atg gct aacatt cga ctt gtg 306 Ala Pro Phe Ile Val Asn Gly Val Glu Met Ala Asn IleArg Leu Val 60 65 70 ggg atg gtc aat gcc aag gtg gag cgg acg acc gat gtgacc ttc acg 354 Gly Met Val Asn Ala Lys Val Glu Arg Thr Thr Asp Val ThrPhe Thr 75 80 85 ctc gac gat ggc acc ggc cgc ctc gat ttc atc aga tgg gtgaat gat 402 Leu Asp Asp Gly Thr Gly Arg Leu Asp Phe Ile Arg Trp Val AsnAsp 90 95 100 gct tca gat tct ttt gaa act gct gct att cag aat ggt atgtac att 450 Ala Ser Asp Ser Phe Glu Thr Ala Ala Ile Gln Asn Gly Met TyrIle 105 110 115 120 gcg gtc att gga agc ctc aag gga ctg caa gag agg aagcgt gct act 498 Ala Val Ile Gly Ser Leu Lys Gly Leu Gln Glu Arg Lys ArgAla Thr 125 130 135 gct ttc tca atc agg cct ata acc gat ttc aat gag gttacg ctg cat 546 Ala Phe Ser Ile Arg Pro Ile Thr Asp Phe Asn Glu Val ThrLeu His 140 145 150 ttc att cag tgt gtt cgg atg cat ata gag aac act gaatta aag gct 594 Phe Ile Gln Cys Val Arg Met His Ile Glu Asn Thr Glu LeuLys Ala 155 160 165 ggc agt cct gca cga atc aat tct tct atg gga gtg tcattc tca aat 642 Gly Ser Pro Ala Arg Ile Asn Ser Ser Met Gly Val Ser PheSer Asn 170 175 180 gga ttc agt gaa tca agc aca ccg aca tct ttg aaa tccagt ccc gca 690 Gly Phe Ser Glu Ser Ser Thr Pro Thr Ser Leu Lys Ser SerPro Ala 185 190 195 200 ccg gtg acc agc ggg tca tcc gat act gat ctg cacacg cag gtc ctg 738 Pro Val Thr Ser Gly Ser Ser Asp Thr Asp Leu His ThrGln Val Leu 205 210 215 aat ttt ttt aat gaa cca gcg aac ctc gag agt gagcat ggg gtg cac 786 Asn Phe Phe Asn Glu Pro Ala Asn Leu Glu Ser Glu HisGly Val His 220 225 230 gtt gat gaa gta ctc aag cgg ttc aac ttt tgc cgaaga agc aga tca 834 Val Asp Glu Val Leu Lys Arg Phe Asn Phe Cys Arg ArgSer Arg Ser 235 240 245 cgg atg cta ttg att aca ata tgg act cgg ggc gtcttt act caa caa 882 Arg Met Leu Leu Ile Thr Ile Trp Thr Arg Gly Val PheThr Gln Gln 250 255 260 ttg atg aat tcc act aca agg caa ctt aac cga tttgaa ggt cag cct 930 Leu Met Asn Ser Thr Thr Arg Gln Leu Asn Arg Phe GluGly Gln Pro 265 270 275 280 gct gga aat ggc aga gga cta agt atc act tgtact aaa cca aag tct 978 Ala Gly Asn Gly Arg Gly Leu Ser Ile Thr Cys ThrLys Pro Lys Ser 285 290 295 gga aat gtc atg ttg tgt cat gaa atg cat ggttgg ttt atg gaa aca 1026 Gly Asn Val Met Leu Cys His Glu Met His Gly TrpPhe Met Glu Thr 300 305 310 ttt ata tct tgt atc aac tagttgatttgtatctcttg tgtcaaaaaa 1074 Phe Ile Ser Cys Ile Asn 315 aaaaaaaaaa aaa1087 18 318 PRT Zea mays VARIANT (1)...(318) Xaa = Any Amino Acid 18 MetMet Pro Leu Ser Gln Thr Asp Phe Ser Pro Ser Gln Phe Thr Ser 1 5 10 15Ser Gln Asn Ala Ala Ala Asp Ser Thr Thr Pro Ser Lys Met Arg Gly 20 25 30Ala Ser Ser Thr Met Pro Leu Thr Val Lys Xaa Val Val Asp Ala Gln 35 40 45Gln Ser Gly Thr Gly Glu Lys Gly Ala Pro Phe Ile Val Asn Gly Val 50 55 60Glu Met Ala Asn Ile Arg Leu Val Gly Met Val Asn Ala Lys Val Glu 65 70 7580 Arg Thr Thr Asp Val Thr Phe Thr Leu Asp Asp Gly Thr Gly Arg Leu 85 9095 Asp Phe Ile Arg Trp Val Asn Asp Ala Ser Asp Ser Phe Glu Thr Ala 100105 110 Ala Ile Gln Asn Gly Met Tyr Ile Ala Val Ile Gly Ser Leu Lys Gly115 120 125 Leu Gln Glu Arg Lys Arg Ala Thr Ala Phe Ser Ile Arg Pro IleThr 130 135 140 Asp Phe Asn Glu Val Thr Leu His Phe Ile Gln Cys Val ArgMet His 145 150 155 160 Ile Glu Asn Thr Glu Leu Lys Ala Gly Ser Pro AlaArg Ile Asn Ser 165 170 175 Ser Met Gly Val Ser Phe Ser Asn Gly Phe SerGlu Ser Ser Thr Pro 180 185 190 Thr Ser Leu Lys Ser Ser Pro Ala Pro ValThr Ser Gly Ser Ser Asp 195 200 205 Thr Asp Leu His Thr Gln Val Leu AsnPhe Phe Asn Glu Pro Ala Asn 210 215 220 Leu Glu Ser Glu His Gly Val HisVal Asp Glu Val Leu Lys Arg Phe 225 230 235 240 Asn Phe Cys Arg Arg SerArg Ser Arg Met Leu Leu Ile Thr Ile Trp 245 250 255 Thr Arg Gly Val PheThr Gln Gln Leu Met Asn Ser Thr Thr Arg Gln 260 265 270 Leu Asn Arg PheGlu Gly Gln Pro Ala Gly Asn Gly Arg Gly Leu Ser 275 280 285 Ile Thr CysThr Lys Pro Lys Ser Gly Asn Val Met Leu Cys His Glu 290 295 300 Met HisGly Trp Phe Met Glu Thr Phe Ile Ser Cys Ile Asn 305 310 315 19 1074 DNAZea mays misc_feature (0)...(0) Maize RPA Middle Subunit Homologue-6 19gacccacgcg tccgcgcaag cctattcgcc gcacctcctc aggtgaccgg gaag atg 57 Met 1atg ccg ttg agc caa acc gac ttc tcg ccg tcg cag ttc acc tcc tcc 105 MetPro Leu Ser Gln Thr Asp Phe Ser Pro Ser Gln Phe Thr Ser Ser 5 10 15 cagaat gcc gcc gcc gac tcc acc acg cct tcc aag atg cgc ggc gcg 153 Gln AsnAla Ala Ala Asp Ser Thr Thr Pro Ser Lys Met Arg Gly Ala 20 25 30 tcc agcacc atg ccg ctc acc gtg aag cag gtc gtc gac gcg cag cag 201 Ser Ser ThrMet Pro Leu Thr Val Lys Gln Val Val Asp Ala Gln Gln 35 40 45 tct ggc acgggc gag aag ggc gct ccg ttc atc gtc aat ggc gtc gag 249 Ser Gly Thr GlyGlu Lys Gly Ala Pro Phe Ile Val Asn Gly Val Glu 50 55 60 65 atg gct aacatt cga ctt gtg ggg atg gtc aat gcc aag gtg gag cgg 297 Met Ala Asn IleArg Leu Val Gly Met Val Asn Ala Lys Val Glu Arg 70 75 80 acg acc gat gtgacc ttc acg ctc gac gat ggc acc ggc cgc ctc gat 345 Thr Thr Asp Val ThrPhe Thr Leu Asp Asp Gly Thr Gly Arg Leu Asp 85 90 95 ttc atc aga tgg gtgaat gat gct tca gat tct ttt gaa act gct gct 393 Phe Ile Arg Trp Val AsnAsp Ala Ser Asp Ser Phe Glu Thr Ala Ala 100 105 110 att cag aat ggt atgtac att gcg gtc att gga agc ctc aag gga ctg 441 Ile Gln Asn Gly Met TyrIle Ala Val Ile Gly Ser Leu Lys Gly Leu 115 120 125 caa gag agg aag cgtgct act gct ttc tca atc agg cct ata acc gat 489 Gln Glu Arg Lys Arg AlaThr Ala Phe Ser Ile Arg Pro Ile Thr Asp 130 135 140 145 ttc aat gag gttacg ctg cat ttc att cag tgt gtt cgg atg cat ata 537 Phe Asn Glu Val ThrLeu His Phe Ile Gln Cys Val Arg Met His Ile 150 155 160 gag aac act gaatta aag gct ggc agt cct gca cga atc aat tct tct 585 Glu Asn Thr Glu LeuLys Ala Gly Ser Pro Ala Arg Ile Asn Ser Ser 165 170 175 atg gga gtg tcattc tca aat gga ttc agt gaa tca agc aca ccg aca 633 Met Gly Val Ser PheSer Asn Gly Phe Ser Glu Ser Ser Thr Pro Thr 180 185 190 tct ttg aaa tccagt ccc gca ccg gtg acc agc ggg tca tcc gat act 681 Ser Leu Lys Ser SerPro Ala Pro Val Thr Ser Gly Ser Ser Asp Thr 195 200 205 gat ctg cac acgcag gtc ctg aat ttt ttt aat gaa cca gcg aac ctc 729 Asp Leu His Thr GlnVal Leu Asn Phe Phe Asn Glu Pro Ala Asn Leu 210 215 220 225 gag agt gagcat ggg gtg cac gtt gat gaa gta ctc aag cgg ttc aaa 777 Glu Ser Glu HisGly Val His Val Asp Glu Val Leu Lys Arg Phe Lys 230 235 240 ctt ttg ccgaag aag cag atc acg gat gct att gat tac aat atg gac 825 Leu Leu Pro LysLys Gln Ile Thr Asp Ala Ile Asp Tyr Asn Met Asp 245 250 255 tcg ggg cgtctt tac tca aca att gat gaa ttc cac tac aag gca act 873 Ser Gly Arg LeuTyr Ser Thr Ile Asp Glu Phe His Tyr Lys Ala Thr 260 265 270 taaccgatttgaaggtcagc ctgctggaaa tggcagagga ctaagtatca cttgtactaa 933 accaaagtctggaaatgtca tgttgtgtca tgaaatgcat ggttggttta tggaaacatt 993 tatatcttgtatcaactagt tgatttgtat ctcttgtgtc aacttaatga ctgagccaac 1053 aaaaggaaaaaaaaaaaaaa a 1074 20 273 PRT Zea mays 20 Met Met Pro Leu Ser Gln Thr AspPhe Ser Pro Ser Gln Phe Thr Ser 1 5 10 15 Ser Gln Asn Ala Ala Ala AspSer Thr Thr Pro Ser Lys Met Arg Gly 20 25 30 Ala Ser Ser Thr Met Pro LeuThr Val Lys Gln Val Val Asp Ala Gln 35 40 45 Gln Ser Gly Thr Gly Glu LysGly Ala Pro Phe Ile Val Asn Gly Val 50 55 60 Glu Met Ala Asn Ile Arg LeuVal Gly Met Val Asn Ala Lys Val Glu 65 70 75 80 Arg Thr Thr Asp Val ThrPhe Thr Leu Asp Asp Gly Thr Gly Arg Leu 85 90 95 Asp Phe Ile Arg Trp ValAsn Asp Ala Ser Asp Ser Phe Glu Thr Ala 100 105 110 Ala Ile Gln Asn GlyMet Tyr Ile Ala Val Ile Gly Ser Leu Lys Gly 115 120 125 Leu Gln Glu ArgLys Arg Ala Thr Ala Phe Ser Ile Arg Pro Ile Thr 130 135 140 Asp Phe AsnGlu Val Thr Leu His Phe Ile Gln Cys Val Arg Met His 145 150 155 160 IleGlu Asn Thr Glu Leu Lys Ala Gly Ser Pro Ala Arg Ile Asn Ser 165 170 175Ser Met Gly Val Ser Phe Ser Asn Gly Phe Ser Glu Ser Ser Thr Pro 180 185190 Thr Ser Leu Lys Ser Ser Pro Ala Pro Val Thr Ser Gly Ser Ser Asp 195200 205 Thr Asp Leu His Thr Gln Val Leu Asn Phe Phe Asn Glu Pro Ala Asn210 215 220 Leu Glu Ser Glu His Gly Val His Val Asp Glu Val Leu Lys ArgPhe 225 230 235 240 Lys Leu Leu Pro Lys Lys Gln Ile Thr Asp Ala Ile AspTyr Asn Met 245 250 255 Asp Ser Gly Arg Leu Tyr Ser Thr Ile Asp Glu PheHis Tyr Lys Ala 260 265 270 Thr 21 1231 DNA Zea mays misc_feature(0)...(0) Maize RPA Middle Subunit Homologue-7 21 tcccgggtcg acccacgcgtccgcgatcct cccatctgcg cacccgcaag cctattcgcc 60 gcacctcctc aggtgaccgggaag atg atg ccg ttg agc caa acc gac ttc 111 Met Met Pro Leu Ser Gln ThrAsp Phe 1 5 tcg ccg tcg cag ttc acc tcc tcc cag aat gcc gcc gcc gac tccacc 159 Ser Pro Ser Gln Phe Thr Ser Ser Gln Asn Ala Ala Ala Asp Ser Thr10 15 20 25 acg cct tcc aag atg cgc ggc gcg tcc agc acc atg ccg ctc accgtg 207 Thr Pro Ser Lys Met Arg Gly Ala Ser Ser Thr Met Pro Leu Thr Val30 35 40 aag cag gtc gtc gac gcg cag cag tct ggc acg ggc gag aag ggc gct255 Lys Gln Val Val Asp Ala Gln Gln Ser Gly Thr Gly Glu Lys Gly Ala 4550 55 ccg ttc atc gtc aat ggc gtc gag atg gct aac att cga ctt gtg ggg303 Pro Phe Ile Val Asn Gly Val Glu Met Ala Asn Ile Arg Leu Val Gly 6065 70 atg gtc aat gcc aag gtg gag cgg acg acc gat gtg acc ttc acg ctc351 Met Val Asn Ala Lys Val Glu Arg Thr Thr Asp Val Thr Phe Thr Leu 7580 85 gac gat ggc acc ggc cgc ctc gat ttc atc aga tgg gtg aat gat gct399 Asp Asp Gly Thr Gly Arg Leu Asp Phe Ile Arg Trp Val Asn Asp Ala 9095 100 105 tca gat tct ttt gaa act gct gct att cag aat ggt atg tac attgcg 447 Ser Asp Ser Phe Glu Thr Ala Ala Ile Gln Asn Gly Met Tyr Ile Ala110 115 120 gtc att gga agc ctc aag gga ctg caa gag agg aag cgt gct actgct 495 Val Ile Gly Ser Leu Lys Gly Leu Gln Glu Arg Lys Arg Ala Thr Ala125 130 135 ttc tca atc agg cct ata acc gat ttc aat gag gtt acg ctg catttc 543 Phe Ser Ile Arg Pro Ile Thr Asp Phe Asn Glu Val Thr Leu His Phe140 145 150 att cag tgt gtt cgg atg cat ata gag aac act gaa tta aag gctggc 591 Ile Gln Cys Val Arg Met His Ile Glu Asn Thr Glu Leu Lys Ala Gly155 160 165 agt cct gca cga atc aat tct tct atg gga gtg tca ttc tca aatgga 639 Ser Pro Ala Arg Ile Asn Ser Ser Met Gly Val Ser Phe Ser Asn Gly170 175 180 185 ttc agt gaa tca agc aca ccg aca tct ttg aaa tcc agt cccgca ccg 687 Phe Ser Glu Ser Ser Thr Pro Thr Ser Leu Lys Ser Ser Pro AlaPro 190 195 200 gtg acc agc ggg tca tcc gat act gat ctg cac acg cag gtcctg aat 735 Val Thr Ser Gly Ser Ser Asp Thr Asp Leu His Thr Gln Val LeuAsn 205 210 215 ttt ttt aat gaa cca gcg aac ctc gag agt gag cat ggg gtgcac gtt 783 Phe Phe Asn Glu Pro Ala Asn Leu Glu Ser Glu His Gly Val HisVal 220 225 230 gat gaa gta ctc aag cgg ttc aaa ctt ttg ccg aag aag cagatc acg 831 Asp Glu Val Leu Lys Arg Phe Lys Leu Leu Pro Lys Lys Gln IleThr 235 240 245 gat gct att gat tac aat atg gac tcg ggg cgt ctt tac tcaaca att 879 Asp Ala Ile Asp Tyr Asn Met Asp Ser Gly Arg Leu Tyr Ser ThrIle 250 255 260 265 gat gaa ttc cac tac aag gca act taaccgatttgaaggtcagc ctgctggaaa 933 Asp Glu Phe His Tyr Lys Ala Thr 270 tggcagaggactaagtatca cttgtactaa accaaagtct ggaaatgtca tgttgtgtca 993 tgaaatgcatggttggttta tggaaacatt tatatcttgt atcaactagt tgatttgtat 1053 ctcttgtgtcaacttaatga ctgagccaac aaaaggaaga tgtagaggca gacagacatt 1113 tgtagattggctgatagctg attcgggtag ctggtccaat tgcaatctgg ggcccaataa 1173 ttcagatgcaaaagcagaaa gatatttcaa aaaaaaaaaa aaaaaaaaaa aaaaaaaa 1231 22 273 PRT Zeamays 22 Met Met Pro Leu Ser Gln Thr Asp Phe Ser Pro Ser Gln Phe Thr Ser1 5 10 15 Ser Gln Asn Ala Ala Ala Asp Ser Thr Thr Pro Ser Lys Met ArgGly 20 25 30 Ala Ser Ser Thr Met Pro Leu Thr Val Lys Gln Val Val Asp AlaGln 35 40 45 Gln Ser Gly Thr Gly Glu Lys Gly Ala Pro Phe Ile Val Asn GlyVal 50 55 60 Glu Met Ala Asn Ile Arg Leu Val Gly Met Val Asn Ala Lys ValGlu 65 70 75 80 Arg Thr Thr Asp Val Thr Phe Thr Leu Asp Asp Gly Thr GlyArg Leu 85 90 95 Asp Phe Ile Arg Trp Val Asn Asp Ala Ser Asp Ser Phe GluThr Ala 100 105 110 Ala Ile Gln Asn Gly Met Tyr Ile Ala Val Ile Gly SerLeu Lys Gly 115 120 125 Leu Gln Glu Arg Lys Arg Ala Thr Ala Phe Ser IleArg Pro Ile Thr 130 135 140 Asp Phe Asn Glu Val Thr Leu His Phe Ile GlnCys Val Arg Met His 145 150 155 160 Ile Glu Asn Thr Glu Leu Lys Ala GlySer Pro Ala Arg Ile Asn Ser 165 170 175 Ser Met Gly Val Ser Phe Ser AsnGly Phe Ser Glu Ser Ser Thr Pro 180 185 190 Thr Ser Leu Lys Ser Ser ProAla Pro Val Thr Ser Gly Ser Ser Asp 195 200 205 Thr Asp Leu His Thr GlnVal Leu Asn Phe Phe Asn Glu Pro Ala Asn 210 215 220 Leu Glu Ser Glu HisGly Val His Val Asp Glu Val Leu Lys Arg Phe 225 230 235 240 Lys Leu LeuPro Lys Lys Gln Ile Thr Asp Ala Ile Asp Tyr Asn Met 245 250 255 Asp SerGly Arg Leu Tyr Ser Thr Ile Asp Glu Phe His Tyr Lys Ala 260 265 270 Thr

What is claimed is:
 1. An isolated polynucleotide comprising a memberselected from the group consisting of: (a) a nucleic acid sequencehaving at least 80% sequence identity over the full-length of SEQ IDNOS: 11, 13, 15, 17, 19, or 21, as determined by the GAP algorithm underdefault parameters, wherein said sequence encodes a polypeptide whichenhances homologous recombination as part of an RPA complex; and (b) anucleic acid sequence which is fully complementary to the nucleic acidsequence of (a).
 2. The isolated polynucleotide of claim 1, wherein thenucleic acid sequence of (a) has at least 85% sequence identity to SEQID NOS: 11, 13, 15, 17, 19, or
 21. 3. The isolated polynucleotide ofclaim 1, wherein the nucleic acid sequence has at least 90% sequenceidentity to SEQ ID NOS: 11, 13, 15, 17, 19, or
 21. 4. The isolatedpolynucleotide of claim 1, wherein the nucleic acid sequence has atleast 95% sequence identity to SEQ ID NOS: 11, 13, 15, 17, 19, or
 21. 5.The isolated polynucleotide of claim 1, wherein the polynucleotide isSEQ ID NOS: 11, 13, 15, 17, 19, or
 21. 6. An isolated polynucleotideencoding a polypeptide of SEQ ID NOS: 12, 14, 16, 18, 20, or 22, whereinthe polypeptide enhances homologous recombination as part of an RPAcomplex.
 7. An expression cassette comprising the polynucleotide ofclaim 1 operably linked to a promoter. 8 A host cell comprising theexpression cassette of claim
 7. 9. A host cell comprising thepolynucleotide of claim
 1. 10. The host cell of claim 9, wherein thehost cell is a plant cell.
 11. The plant cell of claim 10, wherein theplant cell is from a monocot.
 12. The plant cell of claim 11, whereinthe monocot is maize, sorghum, rice, barley, millet, rye, or wheat. 13.The plant cell of claim 10, wherein the plant cell is from a dicot. 14.The plant cell of claim 13, wherein the dicot is soybean, canola,alfalfa, sunflower, or safflower.
 15. A plant comprising the host cellof claim
 10. 16. The plant of claim 15, wherein the plant is a monocot.17. The plant of claim 16, wherein the monocot is maize, sorghum, rice,barley, millet, rye, or wheat.
 18. The plant of claim 15, wherein theplant is a dicot.
 19. The plant of claim 18, wherein the dicot issoybean, canola, alfalfa, sunflower, or safflower.
 20. A seed comprisingthe host cell of claim
 10. 21. The seed of claim 20, wherein the seed isfrom a monocot.
 22. The seed of claim 21, wherein the monocot is maize,sorghum, rice, barley, millet, rye, or wheat.
 23. The seed of claim 20,wherein the seed is from a dicot.
 24. The seed of claim 23, wherein thedicot is soybean, canola, alfalfa, sunflower, or safflower.
 25. Anisolated polynucleotide comprising at least 50 contiguous nucleotides ofSEQ ID NO: 11, 13, 15, 17, 19, or
 21. 26. An isolated polynucleotidecomprising a member selected from the group consisting of: (a) a nucleicacid sequence encoding a polypeptide having at least 90% sequenceidentity over the entire length of SEQ ID NO: 12, 14, 16, 18, 20, or 22,as determined by the GAP algorithm under default parameters, wherein theencoded polypeptide enhances homologous recombination as part of an RPAcomplex; and (b) a nucleic acid sequence which is fully complementary tothe nucleic acid sequence of (a).
 27. The isolated polynucleotide ofclaim 26, wherein the nucleic acid sequence of (a) encodes a polypeptidehaving at least 95% sequence identity to SEQ ID NO: 12, 14, 16, 18, 20,or
 22. 28. The isolated polynucleotide of claim 26, wherein the nucleicacid sequence of (a) encodes the polypeptide of SEQ ID NO: 12, 14, 16,18, 20, or
 22. 29. An isolated polynucleotide comprising a nucleic acidsequence which selectively hybridizes to the full-length complement ofSEQ ID NO: 11, 13, 15, 17, 19, or 21 under high stringency hybridizationconditions and a wash in 0.1×SCC at 60-65° C., wherein saidpolynucleotide encodes a polypeptide which enhances homologousrecombination as part of an RPA complex.
 30. A method for enhancinghomologous recombination in a plant cell, said method comprisingtransforming the plant cell with an expression cassette comprising thepolynucleotide of claim 1 operably linked to a promoter that drivesexpression in the plant cell.
 31. The method of claim 30, wherein theplant cell is from a monocot.
 32. The method of claim 31, wherein themonocot is maize, sorghum, rice, barley, millet, rye, or wheat.
 33. Themethod of claim 30, wherein the plant cell is from a dicot.
 34. Themethod of claim 33, wherein the dicot is soybean, canola, alfalfa,sunflower, or safflower.
 35. A method for increasing pathogen resistancein a plant cell, said method comprising transforming said plant cellwith an expression cassette comprising the polynucleotide of claim 1operably linked in antisense orientation to a pathogen-induciblepromoter.
 36. The method of claim 35, wherein the plant cell is from amonocot.
 37. The method of claim 36, wherein the monocot is maize,sorghum, rice, barley, millet, rye, or wheat.
 38. The method of claim35, wherein the plant cell is from a dicot.
 39. The method of claim 38,wherein the dicot is soybean, canola, alfalfa, sunflower, or safflower.40. A method for modulating DNA metabolism in a plant cell, said methodcomprising transforming said plant cell with an expression cassettecomprising the polynucleotide of claim 1 operably linked to a promoter.41. A method for influencing cell cycle in a plant cell, said methodcomprising transforming said plant cell with an expression cassettecomprising the polynucleotide of claim 1 operably linked to a promoter.